N-3 polyunsaturated fatty acids, especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), mitigate the progression of obesity-associated insulin resistance. However, the knowledge on the metabolic response and underlying mechanism of α-linolenic acid-rich linseed oil (ALA-LO) on insulin resistance was still limited. The results showed that lard-based high fat diet (HFD) feeding for 16 weeks (60% of total calories from fat) led to imbalanced lipid synthesis-oxidation, defect insulin signaling and serious mitochondrial damage in mice liver, which was attenuated by LO. Moreover, LO blocked chronic HFD-induced mitophagy suppression, and improved mitochondrial biogenesis and fusion process in mice liver. Importantly, the specific location of ALA, and its n-3 derivatives docosapentenoic acid (DPA) and DHA in mitochondrial membrane was observed, concomitant with upregulation of SIRT1, PPARγ coactivator-1α (PGC-1α) and extracellular signal regulated kinase (ERK) in mice liver. LO improves hepatic insulin resistance in obese mice partly by restoring mitochondrial quality control network.