My game will require more than one playthrough to understand the story 100%.
Do you think it will be a major put-off for players?
I would like to hear your opinions.
Depends on a vast number of variables.
1. How long is your game? Too long, and I won't want to play it a second time just to see what's different and what more I can learn.
2. How fun is your game? If it's boring, I'll play it once... sometimes not even once, and be done.
3. How interesting is the story? If I don't find it interesting I'm not going to care about seeing it to completion once, much less a second time.
4. How convoluted is the story that I need to play a game twice to see it all? Ideally, you'd want the second playthrough to not answer any questions, but instead elaborate and explain the stuff the player already knows. First playthrough should answer all your questions.
5. How quickly can I get to making the new choices or having the new game to explain the stuff? If I have to wade through another hour of the game just to start getting new information, then I don't care.
6. Are the characters engaging a second time through the game? Most of the time, characters are good for a single play through of a game. Or a book you read. Or a movie you watch. They're rarely good the second time around, unless they've got all new dialogue and scenes the second time around.
7. Is there any reason to play the game a second time for the answers beyond "I'm curious"? If all I am is curious, I might just wait until someone finishes it, posts the ending and all the information, and read their stuff instead of having to slog through it myself.
Personally, I'd concentrate on making a game people want to play through once and enjoy the heck out of.