Lysosomes, the degradative organelle of the endocytic and autophagic pathways, function at an acidic pH, but the molecular processes that determine lysosomal pH are not understood. In particular, no lysosomal pH sensitive signaling enzymes have yet been identified. Here we show genetically and pharmacologically that in the absence of bicarbonate (HCO3-) regulated soluble adenylyl cyclase (sAC), lysosomes fail to properly acidify leading to accumulation of autophagic vacuoles (AVs). Due to the ubiquitous presence of carbonic anhydrases (CAs), which instantaneously equilibrate carbon dioxide (CO2), HCO3-, and intracellular pH (pHi), sAC serves as a physiological sensor of HCO3-, CO2, and pHi. We show that sAC’s role in lysosomal acidification is dependent upon its regulation by intracellular HCO3- in a CA dependent manner, consistent with sAC serving as a pH sensor regulating lysosomal pH. Thus, sAC is the first, and thus far only known, pH regulated signaling enzyme that affects lysosomal pH. Because intracellular vesicles like lysosome are functionally dependent upon acidification, this work has broad implications.