Real have launched an internal inquiry after Di Maria was caught on television briefly grabbing his genitals while being substituted in Monday's La Liga game at home to Celta Vigo. He denied the action was directed at the fans who whistled him off or Ancelotti.
The 25-year-old, replaced by Gareth Bale midway through the second half, insisted it was "a natural gesture" and said he apologised if anyone had found it offensive.
The incident was widely debated in the media and heightened speculation that Di Maria, who has made fewer appearances since the arrival of world record signing Bale, might be sold either in the January transfer window or at the end of the season.
"We have to believe the player," Ancelotti told a news conference previewing Thursday's King's Cup last 16, first leg game at home to Osasuna.
"If anyone thought it was an ugly gesture he has apologised," he added.
"He is focused and I hope he doesn't have any problems (from the fans) in the next match at the Bernabeu.
"He is calm and wants to have a good season. The Di Maria issue needs to be quickly put behind us."
However, Ancelotti revealed that the Argentine, who is known as "the noodle" due to his slight frame, would not be in the starting 11 for Thursday's Cup game and would be replaced by Wales winger Bale.
"Tomorrow he (Di Maria) is not starting because I am going to use a player who is fresher," Ancelotti said.
"We have Bale, who did not play from the start against Celta and he will be there from the beginning tomorrow."
Real's Spain midfielder Xabi Alonso had been left out of the squad as he has not fully recovered from damage sustained to an ear drum last month, Ancelotti said.
"He is training well and things will improve," he said of Alonso, whose control and passing ability have been a key reason for Real's success in recent seasons.
"He is not medically unfit to play but we have to take note of how a player feels in this situation.
"He is not comfortable and he will consult a specialist today to tackle it."