Jeff Bezos, the billionaire founder of Amazon.com, and his wife, MacKenzie, have agreed to donate $2.5 million to help pass a same-sex marriage referendum in Washington State, instantly becoming among the largest financial backers of gay marriage rights in the country.

With the gift, the couple have doubled the money available to the proponents of Referendum 74, which would legalize same-sex marriage in the state by affirming a law that passed the Legislature this year. Courts or lawmakers have declared gay marriage legal in six other states, but backers of such measures have never succeeded at the ballot box.

Proponents of the effort in Washington State called it a game-changing gift that gives them a fighting chance in November.

“To get this from a straight, married couple sends a powerful message that marriage is seen as a fundamental question of fairness,” Zach Silk, the campaign manager for Washington United for Marriage, said Thursday in an interview.

Mr. Bezos, who founded Amazon.com in 1994 in Seattle and remains its president, now tops a growing list of heterosexual business executives who are replacing wealthy gay people as the some of the biggest donors to the movement behind same-sex marriage and equality for gay men and lesbians. Bill Gates and Steven A. Ballmer of Microsoft each gave $100,000 to the referendum campaign, according to its officials.

But with the seven-figure gift, Mr. Bezos — a famously private executive who runs a $48 billion-a-year retail empire — has now set the bar even higher.

Matthew Staver/Bloomberg News

The Bezoses have made the donation as the gay rights movement is encountering both setbacks and achievements. In May, President Obama announced his support for same-sex marriage, saying that after a long evolution he had concluded that “same-sex couples should be able to get married.”

But the push for marriage rights across the country has repeatedly run into well-financed, well-organized opposition. There have been 32 ballot measures that would have legalized or banned same-sex marriage. Opponents of gay marriage have won in all 32, according to Mr. Silk.

That opposition has mobilized in Washington State. The Web site of the group Preserve Marriage Washington says that “the definition of marriage in Washington is under attack” and argues that “if this law goes unchallenged, voters would have no say and marriage would be changed for every person in our state from being the union of one man and one woman to being a genderless institution.”

Same-sex marriage was legalized by Washington’s Legislature in February after a concerted push by Gov. Christine Gregoire, a Democrat. But opponents collected enough signatures to put the legislation to the voters.

Those opposed to Referendum 74 have said they intend to raise as much as $4 million to defeat it and overturn the legislation. Backers of the law have pledged to raise at least $8 million, and had raised about $2.5 million before the gift by Mr. Bezos and his wife.

The Bezoses declined through a public relations representative to be interviewed. But officials of Washington United for Marriage said the size of the gift was stunning.

Mr. Bezos was approached via e-mail on Sunday by Jennifer Cast, one of Amazon’s earliest employees and a lesbian mother of four children who is now a fund-raising chairwoman of the pro-referendum effort.

In her e-mail, sent Sunday evening, Ms. Cast, 50, implored Mr. Bezos to understand the importance of the issue to her and her longtime partner.

“I want to have the right to marry the love of my life and to let my children and grandchildren know their family is honored like a ‘real’ family,” Ms. Cast wrote. “We need help from straight people. To be very frank, we need help from wealthy straight people who care about us and who want to help us win.”

In an interview on Thursday night, Ms. Cast said she had no idea how Mr. Bezos would respond. Though she had worked closely with him when Amazon had only a few dozen employees, she left the company in 2001 and said she had never talked about same-sex marriage with him.

“We were chatting about the biz. We weren’t chatting about our lives,” she said, recalling her time at the company. “I never, ever in my life talked to him about gay marriage.”

In the e-mail, Ms. Cast described in detail the pain she endured as a young adult and the difficulties she faced publicly acknowledging her sexuality. At the end, she pointedly asked him to donate between $100,000 and $200,000 to the referendum cause.

“Jeff, I suspect you support marriage equality,” she wrote. “I beg you not to sit on the sidelines and hope the vote goes our way. Help us make it so.”

She hit “send” and waited.

Two days later, on Tuesday, she received a reply while in a car with her family. Recalling that moment, she said she had to read it out loud twice to make sure she had read it right.

“Jen,” the e-mail said, “this is right for so many reasons. We’re in for $2.5 million. Jeff & MacKenzie.”