The endocannabinoid anandamide is able to interact with the transient receptor potential vanilloid 1 (TRPV1) channels at a molecular level. As yet, endogenously produced anandamide has not been shown to activate TRPV1, but this is of importance to understand the physiological function of this interaction. Here, we show that intracellular Ca²⁺ mobilization via the purinergic receptor agonist ATP, the muscarinic receptor agonist carbachol or the Ca²⁺‐ATPase inhibitor thapsigargin leads to formation of anandamide, and subsequent TRPV1‐dependent Ca²⁺ influx in transfected cells and sensory neurons of rat dorsal root ganglia (DRG). Anandamide metabolism and efflux from the cell tonically limit TRPV1‐mediated Ca²⁺ entry. In DRG neurons, this mechanism was found to lead to TRPV1‐mediated currents that were enhanced by selective blockade of anandamide cellular efflux. Thus, endogenous anandamide is formed on stimulation of metabotropic receptors coupled to the phospholipase C/inositol 1,4,5‐triphosphate pathway and then signals to TRPV1 channels. This novel intracellular function of anandamide may precede its action at cannabinoid receptors, and might be relevant to its control over neurotransmitter release.