Metabolic shifts, among which lactate exchanges, are critical for tumor development. Lactate metabolism involves lactate dehydrogenases LDHA and LDHB: while LDHA is well known to catalyze the reduction of pyruvate to lactate at the last step of glycolysis, little is known about the contribution of LDHB, preferentially promoting lactate oxidation, to cancer cell proliferation. Autophagy is essential for the recycling of macromolecules. This process is tightly regulated, notably by cell metabolism. In this study, we report that LDHB positively regulates autophagy. We first found that LDHB is localized in the lysosome-enriched cell fraction of oxidative tumor cells cultured under normal conditions. We observed a physical interaction of LDHB with autophagic components LC3 and acidic cysteine cathepsins B. Furthermore a specific siRNA against LDHB (siLDHB, leaving LDHA expression unaffected) induced the accumulation of LC3-containing autophagic vesicles and destabilized lysosomes. These responses were observed in the absence of exogenous lactate. Providing the LDHB substrate lactate or the LDHB product pyruvate to siLDHB-transfected cells did not restore lysosomes, suggesting that protons produced during the conversion of NAD+ to NADH + H+ by LDHB, or the protein as a scaffold, are involved in the maintenance of basal lysosomal function and autophagic flux. In fact, siLDHB decreased downstream autophagic proteolysis through an increase in lysosomal pH impairing lysosomal protease maturation and activity. siLDHB mimicked autophagy inhibition by chloroquine: it induced a reduction in the number of mature acidic autolysosomes, and decreased cell proliferation with no additive effects on chloroquine. Based on these findings, we developed the catalytically inactive Nterminal part of LDHB as a dominant-negative polypeptide inhibiting the proliferation of LDHB-expressing tumor cell. Conclusively, our study unravels LDHB as a novel target to inhibit autophagy in cancer cells.