The Mediterranean was cut off from the Atlantic about 5.9 to 5.3 million years ago, during the Messinian Salinity Crisis. In simple terms, that means roughly 6 million years ago.

A bit more context:

  • The separation began around 5.96–5.97 million years ago.
  • The crisis ended when Atlantic water rushed back in around 5.33 million years ago.
  • So the Mediterranean stayed largely isolated for about 600,000 years.

If you want the one-line answer for a post: About 6 million years ago, give or take a few hundred thousand years.