Bcl-2/adenovirus E1B 19-kDa interacting protein (BNIP3), a pro-apoptosis protein regulated by the methylation status of its promoter, has been implicated in inducing autophagy. However, the roles of BNIP3 and BNIP3-induced autophagy under hypoxia remain uncertain in esophageal squamous cell carcinoma (ESCC). Two esophageal squamous cancer cell lines, CAES17 and KYSE140, were selected on the basis of the expression and methylation status of BNIP3 to investigate the features of BNIP3 under hypoxia. Hypoxia increased cell death and the expression of BNIP3, whose promoter status was lower methylation, in a time-dependent manner. BNIP3 knockdown by RNA interference downregulated cell death. These studies demonstrated that the exposure of ESCC cells to hypoxia increased the autophagic punctate distribution of MDC staining and GFP-LC3 and that autophagy rate could be inhibited by BNIP3-siRNA. In addition, under hypoxia, cells transfected with BNIP3-siRNA exhibited a lower apoptosis rate than the control, and the apoptosis induced by BNIP3 exhibited a caspase-independent manner. Furthermore, the administration of the autophagic inhibitor 3-methyladenine (3-MA) could augment BNIP3-induced cell apoptosis and death, suggesting that autophagy plays a protective role under hypoxia. Together, our studies indicated that BNIP3 exerts prodeath effects through the induction of caspase-independent apoptosis under hypoxia in ESCC, though BNIP3-induced autophagy acting as a survival mechanism.