Fe3O4 nanoparticles (Fe3O4 NPs) are widely used in health sciences, including reproductive biotechnology. In reproductive systems, they have been applied in sperm cryopreservation, nano-purification, drug delivery, and toxicity assessments. However, most studies have evaluated Fe3O4 NPs at only a single size and dose, even though their biological effects may vary with size-dependent characteristics. Therefore, this study aimed to investigate how magnetic Fe3O4 NPs of different sizes and concentrations influence the cryopreservation of bull sperm. Initially Fe3O4 NPs with three sizes (∼6, ∼20, and ∼80 nm) were synthesized, and two concentrations (10 and 50 μg/mL) were added to diluted semen samples, supplemented with a commercial extender and cryopreserved. The findings showed that 6 nm Fe3O4 NPs maintained total and progressive motility at levels similar to the control group at both doses, whereas a reduction in both motility parameters was observed only in the Fe20-10 group. No statistically significant differences were found in kinematic parameters, acrosomal, or plasma membrane integrity. Conversely, the 50 μg/mL doses of the 20 and 80 nm groups decreased mitochondrial membrane potential (MMP). Chromatin condensation improved in all experimental groups compared to the control, which correlates with reduced DNA damage across nanoparticle (NP) treatments. Zeta potential measurements demonstrated that surface charge varied with particle size, and microscopic evaluations revealed the accumulation of positively charged Fe3O4 NPs in the head region. Overall, this study showed that Fe3O4 NPs exhibit distinct size-dependent characteristics, underscoring the importance of preliminary trials to determine the optimal NP size for spermatological applications.