Investors betting against Tesla lost more than $1 billion Thursday as the company's shares rallied the most in over four years, according to estimates from financial technology firm S3 Partners.

Tesla stock closed more than 16 percent higher Thursday at $331.90 per share, meaning short sellers have taken over a $1.1 billion mark-to-market loss on their 35.1 million of shares shorted, estimates S3. With the price move Tesla's short interest is now $11.7 billion and remains the most shorted stock in the U.S. market, according to S3's head of predictive analytics Ihor Dusaniwsky.

"Today's $1.1 billion mark-to-market loss has turned a profitable year for short sellers, they were up $276 million in year-to-date mark-to-market profits prior to today's rally, into a loss," Dusaniwsky said in an emailed statement. He added that shorts are now down $831 million in year-to-date mark-to-market losses.

Tesla chief executive Elon Musk has actively criticized and trolled investors short his company's stock this year. Most recently, the CEO bashed Greenlight Capital's David Einhorn's bet against his company after the hedge fund manager said the firm's bet against Tesla "was our second biggest loser" in the most recent quarter.