Efforts to save the historic Separation Tree at Melbourne's Royal Botanical Gardens have failed, according to the garden's director.

The 400-year-old Red River Gum is dying after it was ringbarked by vandals twice in the past five years.

The tree marks the spot where people celebrated Victoria's separation from NSW in 1850.

Vandals first targeted the tree in 2010, cutting off most of the outer bark tissue. It was again ringbarked in 2013.

The tree marks the spot where people celebrated Victoria's separation from NSW in 1850. ( Supplied: Royal Botanic Gardens Melbourne )

Despite numerous grafting attempts, staff have been unable to save it.

Royal Botanic Gardens director, Professor Tim Entwisle, said staff noticed the tree starting to die in November last year.

"It flowered a lot and fruited, which is not actually a good thing for a tree," he said.

"When you see that happen it's often the tree trying to produce lots of seed because it knows it's on the way out.

"In the last couple of months all the leaves are now dead."

Professor Entwisle told 774 ABC Melbourne staff would reduce the canopy in the next few months in the interests of safety and see if anything was still living near the base.

Staff were also keen to ensure something of the tree remained as a legacy.

"We'll take it back to the main trunk. We're thinking that we might leave something there as a link to that separation event and as a link to that tree having been there for 400 years," Professor Entwisle said.

"We'll use the wood I think for something - we haven't thought quite what yet - and I'd be fascinated in the kind of ideas people might have for that.

"But something that I guess creates a legacy as well."