The endocannabinoid system plays protective roles against the growth and the spreading of several types of carcinomas. Because estrogens regulate this system both in physiological states and cancer, in this paper we evaluated its involvement in endometrial carcinoma, a well-known estrogen-dependant tumor. To test whether the endocannabinoid system is expressed in endometrial cancer, tissue samples were collected both from 18 patients undergoing surgical treatment for endometrial adenocarcinoma and 16 healthy age-matched controls, and treated for Western blot and immunohistochemical analysis. Moreover, tissues were dounce homogenized and submitted to endocannabinoid measurement by liquid chromatography-mass spectrometry. To evaluate the physiological role of the endocannabinoid system, a human endometrial cancer cell-line (AN3CA) was used and transiently transfected with a plasmid containing the cDNA for the endocannabinoid receptor CB2. Cells were incubated for 48 h with an agonist (JWH133) (10 μm) or antagonist (SR144528) (1 μm) of CB2 24 h after transfection, and cell proliferation was measured by the 3-[4,5-dimethyltiazol-2yl]-2,5 diphenyltetrazolium bromide formazan assay. In human endometrial carcinoma biopsies the expression of CB2 receptor and the levels of its ligand, 2-arachidonoylglycerol increased, whereas monoacylglyerol lipase, an enzyme responsible for 2-arachidonoylglycerol degradation, was down-regulated. Immunohystochemical analysis revealed that CB2 was overexpressed only in malignant endometrial cells. CB2-overexpressing AN3CA cells showed a significant reduction in cell vitality compared with parental AN3CA cells: incubation with the selective CB2 antagonist SR144128 restored the viability of CB2-overexpressing cells to that of untransfected cells. In conclusion, the endocannabinoid system seems to play an important role in human endometrial carcinoma, and modulation of CB2 activity/expression may account for a tumor-suppressive effect.