I have several, but if I must pick one, it’s A Place Further Than The Universe. Interestingly, it’s simultaneously the happiest, too.
It wasn’t the case at first, but when I rewatched it, I was purely, emotionally devastated.
My kneejerk reaction to the question is “Grave of the Fireflies”.
It really is in a class all its own. I don’t think I could ever watch it again.
I haven’t been able too either, ~17 years later. I honestly feel like it gave me PTSD.