Autophagic impairment is implicated in nonalcoholic fatty liver disease (NAFLD), but the molecular mechanism is unclear. We found that autophagic flux was significantly inhibited in 3 murine models of NAFLD. Interestingly, the number of acidic organelles and the level of mature cathepsin D were reduced, suggesting defective lysosome acidification. Asparagine synthetase (ASNS) was induced by endoplasmic reticulum stress, leading to the generation of asparagine, which inhibited lysosome acidification. Both steatotic- and asparagine-treated hepatocytes showed reduced lysosomal acidity and retention of lysosomal calcium. Knockdown of ASNS in steatotic hepatocytes restored autophagic flux. As a potential biomarker, increased serum p62/sequestosome 1 (SQSTM1) level was an independent risk factor for patients with steatosis and lobular inflammation. Impaired autophagy in NAFLD is elicited by defective lysosome acidification, which is caused by ASNS-induced asparagine synthesis under endoplasmic reticulum stress and subsequent retention of lysosomal calcium. p62/SQSTM1 could be used as a noninvasive biomarker in the diagnosis of NAFLD patients.—Wang, X., Zhang, X., Chu, E. S. H., Chen, X., Kang, W., Wu, F., To, K.-F., Wong, V. W. S., Chan, H. L. Y., Chan, M. T. V., Sung, J. J. Y., Wu, W. K. K., Yu, J. Defective lysosomal clearance of autophagosomes and its clinical implications in nonalcoholic steatohepatitis.