De Niro, 75, and Hightower, 63, are currently "not living together at this time," said the insider.
Another insider told PEOPLE: "Sometimes things don't work out the way you hope or want them to."
Hightower was De Niro's 2nd wife. They dated for 10 years before tying the knot in 1997. Their son Elliott was born in 1998. The couple split in 1999, but the divorce was never finalized.
In 2004 they renewed their vows, and in December 2011, a daughter, Grace, was born via a surrogate.
De Niro is father to six children and a grandfather of four.
De Niro married his first wife, actress Diahnne Abbott, in 1976. He fathered a son, Raphael, a former actor who is now a real estate broker in New York.
De Niro adopted Diahnne's daughter Drena from a previous relationship.
De Niro is best known for his role as young Vito Corleone in the 1974 film The Godfather Part II, for which he won the Academy Award for Best Supporting Actor. He played mob figures in gangster films including Goodfellas (1990), The Untouchables (1987), Heat (1995), and Casino (1995).
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