To address the problem of polyspermy during artificial breeding practices of commercial free-spawning marine bivalve species, a greater understanding of the mechanisms behind polyspermy blocking is required. Therefore, we investigated the roles of the calcium ion (Ca2+) channel and calmodulin (CaM) in fast electrical polyspermy blocking in the commercial bivalve species, Tegillarca granosa, using the specific inhibitors verapamil hydrochloride and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7). The impacts of Ca2+-channel and CaM inhibition on polyspermy rates and oocyte membrane potential were studied microscopically using the fluorescent dyes Hochest 33258 and DiBAC4(3), respectively. The results showed that the inhibition of the Ca2+-channel and CaM led to a significant increase in polyspermy rates, which may be attributed to significant reductions in both amplitude and duration of membrane potential change during the depolarization process. These findings not only demonstrate that the Ca2+-channel and CaM play crucial roles in fast electrical polyspermy blocking, but also indicate that Ca2+ might be essential for the depolarization process of oocytes of T. granosa.