It has been suggested that autophagy plays pathogenetic roles in cerebral ischemia, brain trauma, and neurodegenerative disorders. 3,4-Methylenedioxymethamphetamine (MDMA or ecstasy) is an illicit drug that causes long-term serotonergic neurotoxicity in the brain. Apoptosis and necrosis have been implicated in MDMA-induced neurotoxicity, but the role of autophagy in MDMA-elicited serotonergic toxicity has not been investigated. The present study aimed to examine the contribution of autophagy to neurotoxicity in serotonergic neurons in in vitro and in vivo animal models challenged with MDMA. Here, we demonstrated that in cultured rat serotonergic neurons, MDMA exposure induced LC3B-densely stained autophagosome formation, accompanying by a decrease in neurite outgrowth. Autophagy inhibitor 3-methyladenine (3-MA) significantly attenuated MDMA-induced autophagosome accumulation, and ameliorated MDMA-triggered serotonergic neurite damage and neuron death. In contrast, enhanced autophagy flux by rapamycin or impaired autophagosome clearance by bafilomycin A1 led to more autophagosome accumulation in serotonergic neurons and aggravated neurite degeneration. In addition, MDMA-induced autophagy activation in cultured serotonergic neurons might be mediated by serotonin transporter (SERT). In an in vivo animal model administered MDMA, neuroimaging showed that 3-MA protected the serotonin system against MDMA-induced downregulation of SERT evaluated by animal-PET with 4-[18F]-ADAM, a SERT radioligand. Taken together, our results demonstrated that MDMA triggers upregulation of autophagy in serotonergic neurons, which appears to be detrimental to neuronal growth.