Previous studies demonstrated that superoxide could initiate and amplify LDH-catalyzed hydrogen peroxide production in aqueous phase, but its physiological relevance is unknown. Here we showed that LDHA and LDHB both exhibited hydrogen peroxide-producing activity, which was significantly enhanced by the superoxide generated from the isolated mitochondria from HeLa cells and patients' cholangiocarcinoma specimen. After LDHA or LDHB were knocked out, hydrogen peroxide produced by Hela or 4T1 cancer cells were significantly reduced. Re-expression of LDHA in LDHA-knockout HeLa cells partially restored hydrogen peroxide production. In HeLa and 4T1 cells, LDHA or LDHB knockout or LDH inhibitor FX11 significantly decreased ROS induction by modulators of the mitochondrial electron transfer chain (antimycin, oligomycin, rotenone), hypoxia, and pharmacological ROS inducers piperlogumine (PL) and phenethyl isothiocyanate (PEITC). Moreover, the tumors formed by LDHA or LDHB knockout HeLa or 4T1 cells exhibited a significantly less oxidative state than those formed by control cells. Collectively, we provide a mechanistic understanding of a link between LDH and cellular hydrogen peroxide production or oxidative stress in cancer cells in vitro and in vivo.