Autophagosome-lysosome fusion and autolysosome acidification constitute late stages in the autophagic process essential to maintain functional autophagic flux and cellular homeostasis. These two steps are disrupted through the V-ATPase inhibitor bafilomycin A1, however the mechanisms potentially linking options are unclear. We lately revisited the function of lysosomal acidification in autophagosome-lysosome fusion, utilizing an in vivo approach in Drosophila. By genetically depleting individual subunits from the V-ATPase, we confirmed its role in lysosomal acidification and autophagic cargo degradation. Surprisingly, vesicle fusion continued to be active in V-ATPase-depleted cells, indicating that autophagosome-lysosome fusion and autolysosome acidification are 2 separable processes. In comparison, bafilomycin A1 inhibited both acidification and fusion, in line with its effects in mammalian cells. Together, these results imply this drug inhibits fusion individually of their impact on V-ATPase-mediated acidification. We identified the ER-calcium ATPase Ca-P60A/dSERCA like a novel target of bafilomycin A1. Autophagosome-lysosome fusion was defective in Ca-P60A/dSERCA-depleted cells, and bafilomycin A1 caused a substantial rise in cytosolic calcium concentration and disrupted Ca-P60A/SERCA-mediated fusion. Thus, bafilomycin A1 disrupts autophagic flux by individually inhibiting V-ATPase-dependent acidification and Ca-P60A/SERCA-dependent autophagosome-lysosome fusion.