Activation of cannabinoid receptor type 1 on presynaptic neurons is postulated to suppress neurotransmission by decreasing Ca2+ influx through high voltage-gated Ca2+ channels. However, recent studies suggest that cannabinoids which activate cannabinoid receptor type 1 can increase neurotransmitter release by enhancing Ca2+ influx in vitro. The aim of the present study was to investigate the modulation of intracellular Ca2+ concentration by the cannabinoid receptor type I agonist anandamide, and its underlying mechanisms. Using whole cell voltage-clamp and calcium imaging in cultured trigeminal ganglion neurons, we found that anandamide directly caused Ca2+ influx in a dose-dependent manner, which then triggered an increase of intracellular Ca2+ concentration. The cyclic adenosine and guanosine monophosphate-dependent protein kinase systems, but not the protein kinase C system, were involved in the increased intracellular Ca2+ concentration by anandamide. This result showed that anandamide increased intracellular Ca2+ concentration and inhibited high voltage-gated Ca2+ channels through different signal transduction pathways.