Sunday’s fire was the second time in just over a year that flames have broken out at the animal attraction near Ladybank, Cupar.
Images on social media showed large flames emerging from a wooden building and clouds of black smoke.
Four fire engines were dispatched to the scene at Birnie Field at around 12.30pm on Sunday.
There were no reported casualties, said a spokeswoman for the fire service.
It was reported that fire had damaged a barn, a caravan, and a car, the spokeswoman said, adding that firefighters had now brought the blaze under control but remained on site.
In a post on its Facebook page, a spokesperson for Fife Zoo said the attraction would remain closed to visitors on Monday “to take a break and get ourselves back on track”.
They continued: “The zoo will be back open soon and we hope you can join us in the endeavour of continuing building the best little zoo in Scotland.
“We will happily refund/reschedule any bookings for today and Monday.
“Thank you to all who have and continue to support us in our journey.
“Much love from all Fife Zoo’s staff and animals.”
Last July, the roof of a building caught fire at the zoo, which says it currently houses more than 45 animals from around 15 species including meerkats, lemurs and armadillos.