This study aimed to investigate the effect of supplementation of chitosan nanoparticles (CSNPs) on the capacitation of bovine spermatozoa during the in vitro fertilization process. Hyperactivated motility (HAM) and acrosome reaction (AR) of sperm cells as well as in vitro fertilization and cleavage rates are the main parameters used to estimate the effect of CSNPs on bovine spermatozoa's fertilizing ability. In this study, three different concentrations of CSNPs (10, 20 and 100 μg/mL) were prepared and characterized. Motile spermatozoa were separated from frozen–thawed semen by a swim-up technique and capacitated in Sperm-TALP medium supplemented with heparin only without CSNPs treatment (positive control), heparin + 10 μg/mL CSNPs, heparin + 20 μg/mL CSNPs, heparin + 100 μg/mL CSNPs and the last one served as a negative control tube which supplemented with 10 μg/mL CSNPs without adding heparin. Sperm cells were incubated for 90 min at 39°C in a 5% CO2 incubator and evaluated every 30 min at intervals. Cumulus oophorus complex (COCs) were matured in a 5% CO2 incubator at 39°C and inseminated in vitro with frozen–thawed bull sperm of the above concentrations. The inseminated oocytes were incubated at 39°C in a 5% CO2 incubator for 24 h and then examined for evidence of fertilization. The results of this investigation showed that HAM and AR were best affected by CSNPs at a concentration of 20 μg/mL during an incubation time of 60 min. As time went on, the overall proportion of spermatozoa with progressive motility (PM) decreased across all groups, and a substantially lower value was found at the dose mentioned above. Additionally, the impact of sperm treated with CSNPs on fertilization rate was assessed. The outcomes demonstrated that in comparison to the other concentrations (10 and 100 μg/mL), the positive control and the negative control, the proportion of fertilized oocytes was significantly higher in the CSNPs concentration (20 μg/mL). In conclusion, it could be inferred from this investigation that CSNPs support sperm functions during IVF and can be used for biomedical interventions in bovine spermatozoa. Additionally, a high IVF rate was achieved by using sperm treated with CSNPs as CSNPs enhance sperm capacitation and acrosome reaction.