Vitrification may alter the cytoskeleton (microtubule, meiotic spindle, microfilament, etc.) and the subsequent fertilization rate of in vitro derived bovine oocytes. This study was conducted to evaluate the effect of vitrification and post-thawing incubation periods on the cytoskeleton and fertilization rate of in vitro matured (IVM) bovine oocytes. Following 22 h of IVM, 184 fresh matured oocytes (MO) were immediately fertilized in vitro and served as a control. The remaining MO (1009) were then vitrified by the solid surface vitrification method. Immediately after thawing, MO were incubated in maturation medium in 5% CO2 at 39 Â°C for 0, 30, 60, 90 and 120 min respectively. Following incubation, half of the MO from each vitrified-thawed treatment group (0, 30, 60, 90, and 120 min) was stained with fluorescein isothiocyanate conjugated (FITC) and propidium iodide (PI) to evaluate the microtubule and DNA or spindle under laser-scanning confocal microscopy. The remaining half from the vitrified-thawed MO treatment groups was washed three times in Brackett and Oliphantâ€™s fertilization medium and in vitro fertilized. Cleavage and blastocyst rates were recorded 48 h post-fertilization. Results demonstrated that vitrification damaged MO zona pellucida (ZP), microtubule (MT), meiotic spindle (MS), and caused chromosomal fragmentation. Both the cleavage (84%) and blastocyst rates (50%) of the control group were significantly higher compared to the vitrified-thawed treatment groups. However, extending the incubation period of vitrified MO to 120 min after thawing (prior to fertilization) improved cleavage (65%) and blastocyst (13%) rates 48 h post-fertilization. Fertilizing vitrified MO immediately (0 min group) after thawing resulted in the lowest cleavage (42%) and blastocyst (1.9%) rates. In conclusion, vitrification reduces the subsequent fertilization rate of MO, however, a prolonged post-thawing incubation period (120 min) improves survival, cleavage and blastocyst formation rates, and enhances the reorganization of MOâ€™s cytoskeleton (MT and MS).