Capsaicin has been used as a topical analgesic to treat diverse pain conditions. We investigated the molecular mechanisms that mediate the inhibition of high-voltage-activated calcium channel currents (ICa) using trigeminal ganglion neurons and a heterologous expression system. Capsaicin inhibited ICa in capsaicin-sensitive trigeminal ganglion neurons, but not in capsaicin-insensitive neurons. Single-cell reverse-transcription polymerase chain reaction revealed the expression of TRPV1 only in capsaicin-sensitive neurons. Capsaicin inhibited ICa in transient receptor potential vanilloid-1-expressing C2D7 cells stably expressing human N-type calcium channels, whereas capsaicin failed to inhibit ICa in naïve C2D7 cells with no endogenous transient receptor potential vanilloid-1 expression. Calcium influx via transient receptor potential vanilloid-1 is not likely to play a critical role in capsaicin-induced ICa inhibition in trigeminal ganglion neurons. ICa inhibition might be one of the mechanisms for the analgesic effect of capsaicin.