Human DNA mismatch repair (MMR) recognizes and binds 5-fluorouracil (5FU) incorporated into DNA and triggers a MMR-dependent cell death. Absence of MMR in a patient's colorectal tumor abrogates 5FU's beneficial effects on survival. Changes in the tumor microenvironment like low extracellular pH (pHe) may diminish DNA repair, increasing genomic instability. Here, we explored if low pHe modifies MMR recognition of 5FU, as 5FU can exist in ionized and non-ionized forms depending on pH. We demonstrate that MMR-proficient cells at low pHe show downregulation of hMLH1, whereas expression of TDG and MBD4, known DNA glycosylases for base excision repair (BER) that can remove 5FU from DNA, were unchanged. We show in vitro that 5FU within DNA pairs with adenine (A) at high and low pH (in absence of MMR and BER). Surprisingly, 5FdU:G was repaired to C:G in hMLH1-deficient cells cultured at both low and normal pHe, similar to MMR-proficient cells. Moreover, both hMSH6 and hMSH3, components of hMutSα and hMutSβ, respectively, bound 5FU within DNA (hMSH6>hMSH3) but is influenced by hMLH1. We conclude that an acidic tumor microenvironment triggers downregulation of hMLH1, potentially removing the execution component of MMR for 5FU cytotoxicity, whereas other mechanisms remain stable to implement overall 5FU sensitivity.