Lysosomal membrane permeabilization (LMP) and subsequently impaired autophagosome degradation was induced in HepG2 cells after treatment with perfluorooctane sulfonate (PFOS) for 24 h in our previous studies. We found that treatment of HepG2 cells with PFOS-induced autophagosome formation at earlier stage (6 h) of treatment in this study. The autophagosome formation inhibitor 3-methyladenine (3-MA) was able to relieve PFOS-induced LMP and release of cathepsin D in HepG2 cells. Knockdown of Spinster 1, a lysosomal membrane permease, attenuated PFOS-induced LMP in HepG2 cells. We proposed that Spinster 1 might work as a specific molecule that linked autophagy with LMP. PFOS-induced collapse of mitochondrial transmembrane potential was cathepsin D and autophagy dependent. Addition of 3-MA relieved PFOS-induced apoptosis, which was evidenced by Hoechst assay, AV/PI staining and caspase-3 activity assay. Inhibition of autophagosome formation by Atg5 siRNA attenuated PFOS-induced apoptosis. Treatment of HepG2 cells with PFOS for 24 h impaired mitophagy, as evidenced by an increase of cells with giant mitochondria and impairment of colocalization of PINK1 with light chain 3. In summary, we report that PFOS induces autophagy-dependent apoptosis in HepG2 cells through the lysosomal-mitochondrial axis and impairment of mitophagy, suggesting that autophagy is a primary target for PFOS toxicity. These findings provide new mechanistic insights into PFOS-induced hepatotoxicity.