Spinal cannabinoid receptor 1 (CBR) and purinergic P2X receptors (P2XR) play a critical role in the process of pathological pain. Both CBR and P2XR are expressed in spinal dorsal horn (DH) neurons. It is not clear whether CB receptor activation modulates the function of P2X receptor channels within dorsal horn. For this reason, we observed the effect of CP55940 (cannabinoid receptor agonist) on ATP-induced Ca mobilization in cultured rat DH neurons. The changes of intracellular calcium concentration ([Ca]i) were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescent indicator. 100 μM ATP caused [Ca]i increase in cultured DH neurons. ATP-evoked [Ca]i increase in DH neurons was blocked by chelating extracellular Ca and P2 purinoceptor antagonist PPADS. At the same time, ATP-γ-S (a non-hydrolyzable ATP analogue) mimicked the ATP action, while P2Y receptor agonist ADP failed to evoke [Ca]i increase in cultured DH neurons. These data suggest that ATP-induced [Ca]i elevation in cultured DH neurons is mediated by P2X receptor. Subsequently, we noticed that, in cultured rat DH neurons, ATP-induced Ca mobilization was inhibited after pretreated with CP55940 with a concentration-dependent manner, which implies that the opening of P2X receptor channels are down-regulated by activation of cannabinoid receptor. The inhibitory effect of CP55940 on ATP-induced Ca response was mimicked by ACEA (CBR agonist), but was not influenced by AM1241 (CBR agonist). Moreover, the inhibitory effect of CP55940 on ATP-induced Ca mobilization was blocked by AM251 (CB receptor antagonist), but was not influenced by AM630 (CB receptor antagonist). In addition, we also observed that forskolin (an activator of adenylate cyclase) and 8-Br-cAMP (a cell-permeable cAMP analog) reversed the inhibitory effect of CP55940, respectively. In a summary, our observations raise a possibility that CBR rather than CBR can downregulate the opening of P2X receptor channels in DH neurons. The reduction of cAMP/PKA signaling is a key element in the inhibitory effect of CBR on P2X-channel-induced Ca mobilization.