Cholangiocarcinomas are devastating cancers of biliary origin with limited treatment options. It has previously been shown that the endocannabinoid anandamide exerts antiproliferative effects on cholangiocarcinoma independent of any known cannabinoid receptors, and by the stabilization of lipid rafts, thereby allowing the recruitment and activation of the Fas death receptor complex. Recently, GPR55 was identified as a putative cannabinoid receptor; therefore, the role of GPR55 in the antiproliferative effects of anandamide was evaluated. GPR55 is expressed in all cholangiocarcinoma cells and liver biopsy samples to a similar level as in non-malignant cholangiocytes. Treatment with either anandamide or the GPR55 agonist, O-1602, reduced cholangiocarcinoma cell proliferation in vitro and in vivo. Furthermore, knocking down the expression of GPR55 prevented the antiproliferative effects of anandamide. Coupled to these effects was an increase in JNK activity. The antiproliferative effects of anandamide could be blocked by pretreatment with a JNK inhibitor and the lipid raft disruptors β-methylcyclodextrin and fillipin III. Activation of GPR55 by anandamide or O-1602 increased the amount of Fas in the lipid raft fractions, which could be blocked by pretreatment with the JNK inhibitor. These data represent the first evidence that GPR55 activation by anandamide can lead to the recruitment and activation of the Fas death receptor complex and that targeting GPR55 activation may be a viable option for the development of therapeutic strategies to treat cholangiocarcinoma.