Nobiletin (NBT) is a unique flavonoid mainly found in citrus fruits, and has been reported to inhibit colon carcinogenesis in multiple rodent models. However, the direct molecular targets of NBT are unknown. Heat shock cognate 70 protein (HSC70) contributes to cancer cell survival and resistance to chemotherapies, thereby the inhibition of HSC70 is a promising strategy in cancer chemoprevention. Using affinity chromatography, proteomics analysis and computer modeling, we demonstrated that NBT bound to HSC70 at its ATP-binding site and specifically inhibited its ATPase activity. The association between HSC70 and HSP90 is critical for the stability of their client proteins, which contributes to carcinogenesis. Our results showed that NBT disrupted the association between HSC70 and HSP90. Moreover, combined treatment with NBT and HSP90 inhibitors led to enhanced inhibition of cancer cell in both cell culture model and mouse model. Interestingly, our results also demonstrated that the major colonic metabolites of NBT (generated in the colon of NBT-fed mice) showed similar or stronger inhibitory effects against HSC70-mediated pro-carcinogenic in comparison with NBT.