Accumulating evidence highlights the importance of the endocannabinoid anandamide as a key mediator in reproductive physiology. Current data suggest potential roles for anandamide in gametogenesis, fertilization and parturition. Anandamide exerts its actions through two G protein-coupled receptors, termed cannabinoid receptor 1 (CB1), and 2 (CB2), and the ligand-gated transient receptor potential vanilloid receptor type 1 (TRPV1) ion channel. At present, the cellular mechanism(s) and consequences of anandamide signaling in reproductive tissues, especially the myometrium, are poorly understood. Here, we examine the expression of CB1, CB2 and TRPV1 in the human myometrial smooth muscle cell-line (ULTR) and characterize intracellular signaling following stimulation with anandamide. Radioligand binding analysis revealed a total CB receptor expression of 76 ± 24 fmol/mg protein, with both quantitative PCR and competition binding studies indicating a negligible CB2 component. Anandamide caused G_i/o-dependent inhibition of adenylate cyclase to reduce intracellular cyclic AMP levels. In addition, anandamide caused a 2.5–3.5-fold increase in ERK activation, which was ablated by inhibition of G_i/o, phosphoinositide-3-kinase and Src-kinase activities, but not by inhibition of Ca²⁺/calmodulin-dependent protein kinase or protein kinase C activities. TRPV1 channel activation with capsaicin failed to activate ERK. Consistent with these findings the selective agonists, arachidonyl-2-chloroethylamide (CB1) and L759656 (CB2), and selective antagonists AM251 (CB1) and JTE907 (CB2), provided pharmacological evidence that the ERK signaling pathway is activated through endogenously expressed CB1. These findings provide an insight into myometrial anandamide signaling, highlighting a potential role for endocannabinoids in the regulation of gene expression in myometrial smooth muscle cells.