The molecular basis for the control of energy balance by the endocannabinoid anandamide (AEA) is still unclear. Here, we show that murine 3T3-L1 fibroblasts have the machinery to bind, synthesize and degrade AEA, and that their differentiation into adipocytes increases by approximately twofold the binding efficiency of cannabinoid receptors (CBR), and by approximately twofold and approximately threefold, respectively, the catalytic efficiency of the AEA transporter and AEA hydrolase. In contrast, the activity of the AEA synthetase and the binding efficiency of vanilloid receptor were not affected by the differentiation process. In addition, we demonstrate that AEA increases by approximately twofold insulin-stimulated glucose uptake in differentiated adipocytes, according to a CB1R-dependent mechanism that involves nitric oxide synthase, but not lipoxygenase or cyclooxygenase. We also show that AEA binding to peroxisome proliferator-activated receptor-gamma, known to induce differentiation of 3T3-L1 fibroblasts into adipocytes, is not involved in the stimulation of glucose uptake.