Background <p>Mitophagy is an essential cellular autophagic process which maintains mitochondrial homeostasis, but its role in high fat diet (HFD)-induced lipid accumulation is unclear in the yellow catfish. Thus, this study aimed to elucidate mechanism of mitochondria mediating HFD-induced hepatic fat accumulation.</p> Results <p>In the present study, yellow catfish were fed three diets with dietary fat at 6.31% (low fat; LFD, control), 12.03% (middle fat; MFD) and 15.32% (high fat; HFD), respectively, for 8&#xa0;weeks. High dietary fat addition raised hepatic lipid accumulation, and declined mRNA and protein levels of Parkin-dependent mitophagy, down-regulated the Parkin protein expression and the estrogen-related receptor alpha (Errα) ubiquitination, and induced Errα protein levels; fatty acid (FA) incubation reduced Parkin-dependent mitophagy, inhibited Errα ubiquitination and increased Errα protein expression, and raised TG accumulation. Furthermore, yellow catfish hepatocytes were isolated and cultured. Nicotinamide mononucleotide, <i>N</i>-acetyl-L-cysteine, Parkin and <i>errα</i> siRNA knockdown were used under FA incubation, respectively. Parkin downregulation mediated FA incubation-induced TG accumulation and mitoautophagic inhibition; Parkin ubiquitinated Errα, and K63 was an important ubiquitination site for deubiquitinating Parkin activity; Errα targets <i>fas</i>, <i>acca</i> and <i>pparγ</i> genes, whose activation contributed to FA-induced lipogenesis and lipid accumulation. Thus, high fat diet (HFD) and FA incubation inhibited Parkin activity, suppressed mitophagy and activated Errα pathway, and induced hepatic lipogenic metabolism and lipotoxicity.</p> Conclusions <p>Overall, our study provided new targets against HFD-induced hepatic lipid accumulation and non-alcoholic fatty liver disease in the vertebrates.</p>