Obesity is associated with skeletal muscle mass loss and physical dysfunction. Krill oil (KO) has been shown to be beneficial in human health. However, the effect of KO on obesity-induced skeletal muscle atrophy is still unclear. In this study, the male C57BL/6J mice were fed a high-fat diet (HFD) for 12 weeks to induce obesity, and then were intragastric administration with 400 mg/kg bw KO for an additional 6 weeks. The results showed that KO treatment reduced body weight, fat accumulation and serum pro-inflammatory cytokines in HFD-induced obese mice. Importantly, KO treatment attenuated skeletal muscle atrophy in HFD-fed mice, as evidenced by preserving skeletal muscle mass, average myofiber cross-sectional area and grip strength. KO administration also mitigated obesity-induced ectopic lipid deposition and inflammatory response in skeletal muscle. Additionally, KO treatment inhibited the transcriptional activities of nuclear factor-κB (NF-κB) p65 and forkhead box O 3a (FoxO3a), and then down-regulated muscle atrophy F-box (MAFbx) and muscle-specific RING finger protein 1 (MuRF1) protein levels in skeletal muscle from HFD-fed mice. KO administration also improved obesity-induced impaired muscle protein synthesis via activating PI3K/Akt pathway. Furthermore, KO treatment enhanced muscle mitochondrial biogenesis in HFD-induced obese mice via activating PGC-1α pathway. Collectively, KO might be developed as a potential nutritional supplement for the prevention and treatment of obesity-induced skeletal muscle atrophy.