Semen preservation is a main limitation in rabbit artificial insemination. The aim of this study was to investigate the effect of gelatin addition at different levels on the quality and fertility of rabbit semen preserved at room (20-25 ^◦C) or refrigerator (5 ◦C) temperature degrees. Semen was collected from 15 APRI bucks aged 1 year with artificial vagina twice/week for 10 weeks. Immediately after collection, ejaculates with 70% or more mass motility were pooled and diluted at a rate of 1:5 in Tris-buffer extender supplemented with 0, 1, 2 and 3% gelatin and each level was divided into two portions, then semen was preserved at room temperature (20-25 ◦C) for the 1^st portions and at 5 ◦C for the 2^nd ones. Motility, livability and acrosome integrity were evaluated in preserved semen at 0, 6, 12, 24, 48 and 72 hours. Synchronized lacting dose were AI with on day 11 post-partum. Results showed that adding gelatin at level of 2% to Tris-extender for both semen preserved at 20-25 or 5 ◦C increased (P<0.05) percentages of progressive motility, livability and acrosome integrity, and decreased abnormality of spermatozoa as compared to the control and other extenders, and spermatozoa kept their qualitative characteristics for 24 h after collection, in order to be used in artificial insemination to receptive does Percentage of motility, livability and acrosome integrity of spermatozoa decreased (P<0.05) and sperm abnormality increased (P<0.05) by advancing storage time in both semen preserved at 20-25 or 5 ◦C. Does inseminated with fresh semen diluted with Tris-buffer extender supplemented with 2% gelatin yielded the highest kindling rate and litter size. In conclusion, rabbit semen could be preserved at room temperature or at 5 ◦C without affecting its quality by adding 2% of gelatin to Tris-buffer extender to avoid expensive materials, and having an easy way to manage and transport within 24 hours.