The decrease of ASPP1 (Apoptosis-Stimulating Protein of p53 1), a known p53 activator, has been linked to carcinogenesis and the cytotoxic resistance in various cancers, yet the underlying mechanisms of ASPP1 expression and its complex functions are not yet clear. Here, we report that ASPP1 forms an inter-regulatory loop with Early Growth Response 1 (EGR-1), and promotes apoptosis via inhibiting cyto-protective autophagy, independent of the well-documented p53-dependent mechanisms. We show that ASPP1 mRNA and protein were remarkably elevated by ectopic EGR-1 expression or endogenous EGR-1 activation, in cells with different tissue origins and p53 status. Conversely, RNAi-mediated EGR-1 knockdown suppressed ASPP1. The further mechanism studies revealed that ASPP1 promoter, mapped to -283/+88, which contained three conserved EGR-1 binding sites, was required for both binding and transactivity of EGR-1. In addition, we demonstrate that ASPP1 promoted EGR-1 in a positive feedback loop by preventing proteasome-mediated EGR-1 degradation or promoting EGR-1 nuclear import in response to anticancer natural compound Quercetin. Furthermore, albeit activating p53 in the nucleus is the well-studied function of ASPP1, we found that ASPP1 was predominately localized in the cytoplasm. Interestingly, the cytoplasmic ASPP1 retained its pro-apoptosis capability. Mechanistically, ASPP1 suppressed Atg5-Atg12 and also bound with Atg5-Atg12 to prevent its further complex formation with Atg16, resulting in the inhibition of cyto-protective autophagy. In conclusion, our results provide new insights into EGR-1/ASPP1 regulatory loop in sensitizing Quercetin-induced apoptosis. EGR-1/ASPP1, therefore, may be potentially used as therapeutic targets to improve cancer's response to pro-apoptosis treatments.