In MCF-7 breast cancer cells, transmembrane prostatic acid phosphatase (TM-PAP) plays a critical role in tumor progression, particularly under hypoxic conditions. In this study, the impact of hypoxia on ectophosphatase activity in MCF-7 cells was examined, and the underlying biological mechanisms that influence the breast cancer microenvironment were explored. Compared with normoxic cells, hypoxic cells presented significant reductions in ectophosphatase activity, indicating that hypoxia altered dephosphorylation processes critical for tumor growth and metastasis. Specific decreases in the hydrolysis of substrates, such as p-nitrophenylphosphate (pNPP) and adenosine monophosphate (AMP), were observed under hypoxic conditions, suggesting that hypoxia impaired TM-PAP activity. Further investigation revealed that hypoxia induced an increase in the concentration of reactive oxygen species (ROS), such as hydrogen peroxide (H