Social sources have noticed that "Raging Bull" star De Niro has been solo at events he'd usually attend with Hightower over the past months.
A source close to the former couple told Page Six on Tuesday that De Niro, 75, and Hightower, 63, are "not living together at this time".
The "Goodfellas" star is said to have split from his spouse and they are expected to make a formal announcement in the coming weeks.
De Niro and Hightower married in 1997 after dating for almost a decade, and they have two kids together. While De Niro has kept his private life out of the spotlight he did show his softer side while speaking about his family.
Hightower is De Niro's second wife.
The couple Wednesday in 1997 and have two children - 20-year-old son Elliott and 6-year-old daughter Helen Grace. Robert filed for divorce in 1999, two years into their union, but they never went all the way through with it. They later reunited and renewed their wedding vows in 2004.
De Niro was previously married to Diahnne Abbott, from 1976 to 1988.
The Mean Streets star is also father to two twin boys, Aaron and Julian with ex girlfriend, USA actress and model, Toukie Smith.
A rep for De Niro did not immediately respond to Fox News' request for comment.
'There are many people who will come out and say I saw my kid change overnight.
De Niro spoke about his marriage to Hightower with journalist Katie Couric in 2013, during which interview he said that marriage involves "a lot of give and take, standing your ground, because then you keep the respect you started out with".