La Liga leaders Real had reeled off an incredible 22 consecutive competitive victories before their trip to Mestalla, but were beaten 2-1 in their first fixture of 2015 after a superb display from the hosts.
Ancelotti's side were put under intense pressure from first whistle to last as Valencia were roared on by a partisan home crowd, and goals from Antonio Barragan and Nicolas Otamendi overturned Cristiano Ronaldo's early penalty to give Nuno Espirito Santo's men a well-deserved three points.
Real will lose top spot in the table if Barcelona win at Real Sociedad later on Sunday, but Ancelotti refused to press the panic button after seeing his team beaten for the first time since early September.
The Italian instead insisted Real's winning run could not last forever and commended Valencia for their excellent performance.
"We had a good game and didn't deserve to lose ... [but] in football, anything can happen," Ancelotti said.
"It's a defeat which we eventually had to suffer, but let's not forget what we've achieved already.
"Congratulations to Valencia - they fought hard."
Ancelotti went on to confirm that he will look to play a full-strength side in Wednesday's Copa del Rey last-16 first leg at neighbours Atletico Madrid.
"We have to put the best possible team out to win on Wednesday," he added.
"Physically the team have been good. The season is long and we're well positioned.
"The defeat doesn't affect us. We'll put what we've done so well behind us."
