Drug resistance has become one of the largest challenges for cancer chemotherapies. Under certain conditions, cancer cells hijack autophagy to cope with therapeutic stress, which largely undermines the chemo-therapeutic efficacy. Currently, biomarkers indicative of autophagy-derived drug resistance remain largely inclusive. Here, we report a novel role of lipid rafts/cholesterol-enriched membrane micro-domains (CEMMs) in autophagosome biogenesis and doxorubicin resistance in breast tumors. We showed that CEMMs are required for the interaction of VAMP3 with syntaxin 6 (STX6, a cholesterol-binding SNARE protein). Upon disruption of CEMM, VAMP3 is released from STX6, resulting in the trafficking of ATG16L1-containing vesicles to recycling endosomes and subsequent autophagosome biogenesis. Furthermore, we found that CEMM marker CAV1 is decreased in breast cancer patients and that the CEMM deficiency-induced autophagy is related to doxorubicin resistance, which is overcome by autophagy inhibition. Taken together, we propose a novel model whereby CEMMs in recycling endosomes support the VAMP3 and STX6 interaction and function as barriers to limit the activity of VAMP3 in autophagic vesicle fusion, thus CEMM deficiency promotes autophagosome biogenesis and doxorubicin resistance in breast tumors.