Methylenedioxymethamphetamine (MDMA) is a psychostimulant with high abuse potential and severe neurotoxicity. According to our previous study, MDMA promotes autophagosome accumulation and contributes to cell death in cultured cortical and serotonergic neurons. However, the detailed mechanism underlying autophagy dysfunction remains unclear. Lysosomes play an important role in autophagic degradation. The present study aimed to examine the role of lysosomal function in autophagic flux in neuronal cultures exposed to MDMA. We showed that MDMA induced enlarged vesicles that accumulate in SH-SY5Y neuroblastoma cells. In addition, we demonstrated that MDMA stimulated dynamin-dependent but clathrin-independent endocytosis, which might contribute to vacuole expansion. Morphological and Western blot analyses revealed that MDMA induced lysosomal swelling, whereas the activity of the lysosomal hydrolytic enzymes cathepsin B and cathepsin D was decreased in SH-SY5Y and cultured cortical neurons, which might lead to autophagosome accumulation and autophagic degradation blockage. Intriguingly, inactivation of cathepsins B and D led to cell death and autophagy-lysosomal dysregulation, which mimicked MDMA-induced neurotoxicity. Consequently, impairment of lysosomal proteolysis and blockage of autophagy degradation contributed to MDMA-induced neurotoxicity in neuronal cultures.