The base excision repair (BER) pathway is required to repair endogenous and exogenous oxidative DNA damage. Multiple DNA repair pathways have been shown to be down-regulated in the tumor microenvironment, whereas APE1/Ref1, a central protein in BER, is overexpressed in many types of solid tumors. APE1/Ref1 has dual functions, participating both in BER and redox regulation of oxidized transcription factors. Here, we show that inhibition of the BER pathway in an acidic tumor microenvironment increases oxidative DNA damage temporally related to increased intracellular reactive oxygen species. Unrepaired oxidative DNA damage results in cell cycle arrests and increased DNA double-strand breaks, leading to cell death. Therefore, up-regulation of BER in solid cancers may represent an adaptive survival response. Consequently, BER inhibition may confer tumor microenvironment targeted cytotoxicity in human cancers. Our data suggest that BER inhibition is a rational basis for cancer therapy with or without other cytotoxic therapy. Additionally, our results offer insight as to why APE1/Ref1 retains its unique dual functionality, both of which counteract environmental oxidative stress.