For successful fertilization mammalian spermatozoa must undergo the acrosome reaction (AR), an exocytotic event that allows this cell to penetrate the outer layer of the oocyte, the zona pellucida (ZP). Four glycoproteins (ZP1–ZP4) compose the human ZP, being ZP3 the physiological inductor of the AR. This process requires changes in intracellular Ca2+ concentration ([Ca2+]i) involving not fully understood mechanisms. Even in mouse sperm, the pharmacologically documented participation of voltage-gated Ca2+ (CaV) channels and store-operated channels (SOCs) in the ZP-induced AR is being debated. The situation in human sperm is even less clear due to the limited availability of human ZP. Here, we used recombinant human ZP3 (rhZP3) produced in baculovirus-infected Sf9 cells to investigate the involvement of CaV channels in the human sperm AR. Our findings showed that Ni2+ and mibefradil at concentrations that block T-type or CaV3 channels, and nimodipine and diltiazem that block L-type or CaV1 channels, significantly inhibited the rhZP3-initiated AR. On the other hand, the AR was insensitive to concentrations of ω-Agatoxin IVA, ω-Conotoxin GVIA and SNX-482 that block P/Q, N and R-type channels, respectively (CaV2 channels). Our overall findings suggest that CaV1 and CaV3 channels participate in human sperm AR. Consistent with this, we detected in human sperm transcripts for the CaV1 auxiliary subunits, α2δ, β1, β2 and β4, but not the neuronal specific isoforms β3 and γ2.