This study aimed to evaluate the effect of adding insulin hormone at different levels (0, 5, 10 and 15 µg/ml) to maturation medium with or without exogenous hormones (FSH, LH and E17β) on the in vitro maturation (IVM) of rabbit oocytes. Total of 20 New Zealand White (NZW) rabbit does (5.5-6 mo of age and 2.5 - 3 kg LBW) were used as oocyte donors. Oocytes were recovered from ovaries of slaughtered does using slicing technique. All oocytes with evenly granulated dark ooplasmwere matured in TCM-199 supplemented with 6% bovine serum albumin. Eight types of TCM-199, four types without and other four with exogenous hormones supplemented with insulin (0, 5, 10 and 15 µg /ml) were used. Oocytes were fixed and stained for examinationafter 18 h at 38.5°C, 5% CO2 and high humidity as a maturation period. Results showed significant (P<0.05) effect of insulin supplementation on IVM of rabbit oocyte only in terms of oocytes reaching MI, TI+MII and degenerated ones. Percentage of mature oocytes reaching MII was improved by all insulin levels as compared to un-supplemented media (42.1-44.6 vs. 34.1%), but the differences were not significant. Percentage of oocytes reaching both TI+MII increased (P<0.05) with insulin supplementation at a level of 5 µg/ml showing the best (P<0.05) improvement on oocyte maturation (51.0%) as compared to other insulin levels (45.5-47.6%) or the control medium (39.5%). Nuclear maturation of rabbit oocytes was not affected significantly when FSH, LH and E2 were deleted from the maturation medium, but Percentage of oocytes reaching both T1+MII stages was enhanced in the presence of hormones during the maturation period regardless of whether oocytes were treated with insulin or not. Maturation rate in term of oocytes reaching T1+MII was affected significantly (P<0.05) by the interaction between insulin and hormonal addition, reflecting improved percentage of oocytes reaching both T1+MII stages by addition of all insulin levels to hormone-TCM-199 medium. In this respect, insulin addition at a level of 5µg/ml showed the best result (58.7%). In conclusion, the present study demonstrated that the supplementation of the maturation medium with insulin improves the in vitro maturation rate of rabbit oocytes when oocytes are maturated in a defined maturation medium with or without hormones (FSH, LH and E2).