Emerging evidence suggests that mitochondrial dysfunction mediates the pathogenesis for non-alcoholic fatty liver disease (NAFLD). Hydroxytyrosol (HT) is a key component of extra virgin olive oil which can exert beneficial effects on NAFLD through modulating mitochondria. However, the mechanism of the impacts of HT still remains elusive. Thus, an in vivo and a series of in vitro experiments were carried out to examine the impacts of hydroxytyrosol (HT) on lipid metabolism and mitochondrial function in fish. For the in vivo experiment, two diets were produced to contain 10% and 16% fat as normal-fat and high-fat diets (NFD and HFD) and two additional diets were prepared by supplementing 200 mg/kg of HT to the NFD and HFD. The test diets were fed to triplicate groups of spotted seabass (Lateolabrax maculatus) juveniles for 8 weeks. The results showed that feeding HFD leads to increased fat deposition in the liver and induces oxidative stress, both of which were ameliorated by HT application. Furthermore, transmission electron microscopy revealed that HFD destroyed mitochondrial cristae and matrix and induced severe hydropic phenotype, while HT administration relieved these alterations. The results of in vitro studies using zebrafish liver cell line (ZFL) showed that HT promotes mitochondrial function and activates PINK1-mediated mitophagy. These beneficial effects of HT disappeared when the cells were treated with cyclosporin A (Csa) as a mitophagy inhibitor. Moreover, the PINK1-mediated mitophagy activation by HT was blocked when compound C (CC) was used as an AMPK inhibitor. In conclusion, our findings demonstrated that HT alleviates fat accumulation, oxidative stress and mitochondrial dysfunction, and its effects are deemed to be mediated via activating mitophagy through the AMPK/PINK1 pathway.