Although inhibitors of the enzymatic hydrolysis of the endocannabinoid 2-arachidonoylglycerol are available, they are either rather weak in vitro (IC 50 > 30 μM) or their selectivity towards other proteins of the endocannabinoid system has not been tested. Here we describe the synthesis and activity in vitro and in vivo of a tetrahydrolipstatin analogue, OMDM169, as a potent inhibitor of 2-AG hydrolysis, capable of enhancing 2-AG levels and of exerting analgesic activity via indirect activation of cannabinoid receptors. OMDM169 exhibited 0.13 μM < IC 50 < 0.41 μM towards 2-AG hydrolysing activities in COS-7 cells and rat cerebellum, and inhibited (IC 50 = 0.89 μM) the human recombinant MAGL, whilst being inactive ( K i > 10 μM) at human CB 1 and CB 2 receptors. However, OMDM169 shared with tetrahydrolipstatin the capability of inhibiting the human pancreatic lipase (IC 50 = 0.6 μM). OMDM169 inhibited fatty acid amide hydrolase and diacylglycerol lipase only at higher concentrations (IC 50 = 3.0 and 2.8 μM, respectively), and, accordingly, it increased by ∼ 1.6-fold the levels of 2-AG, but not anandamide, in intact ionomycin-stimulated N18TG2 neuroblastoma cells. Acute intraperitoneal (i.p.) administration of OMDM169 to mice inhibited the second phase of the formalin-induced nocifensive response with an IC 50 of ∼ 2.5 mg/kg, and concomitantly elevated 2-AG, but not anandamide, levels in the ipsilateral paw of formalin-treated mice. The antinociceptive effect of OMDM169 was antagonized by antagonists of CB 1 and CB 2 receptors, AM251 and AM630, respectively (1 mg/kg, i.p.). OMDM69 might represent a template for the development of selective and even more potent inhibitors of 2-AG hydrolysis.