MeIQx (2-amino-3, 8-dimethylimidazo[4, 5-f]quinoxaline), one of the most abundant heterocyclic aromatic amines (HAAs) found in human diet, is primarily produced during high temperature meat or fish cooking. While MeIQx has been investigated as a potential carcinogen, the cytotoxicity and related molecular mechanisms remain unclear. Here we demonstrate that autophagosome maturation is blocked by MeIQx. Mechanistically, MeIQx inhibits acidification of lysosomes rather than prevents autophagosome-lysosome fusion. Moreover, cellular lipid profiles are altered by MeIQx treatment. Notably, many phospholipids and sphingolipids are significantly up-regulated after exposure to MeIQx. Furthermore, MeIQx decreases expression of pluripotency-associated proteins in mouse embryonic stem cells (ESCs). Together, MeIQx blocks autophagosome maturation through inhibiting acidification of lysosomes, alters lipid metabolism and decreases expression of pluripotent factors. Our studies provide more cytotoxic evidence and elucidate related mechanisms on the risk of HAAs exposure and are expected to promote supervision of food safety and human health.