Because oxidative stress promotes insulin resistance in obesity and type 2 diabetes, it is crucial to find effective antioxidant for the purpose of decreasing this threat. In this study, we explored the effect of astaxanthin, a carotenoid antioxidant, on insulin signaling and investigated whether astaxanthin improves cytokine- and free fatty acid-induced insulin resistance in vitro. We examined the effect of astaxanthin on insulin-stimulated glucose transporter 4 (GLUT4) translocation, glucose uptake, and insulin signaling in cultured rat L6 muscle cells using plasma membrane lawn assay, 2-deoxyglucose uptake, and Western blot analysis. Next, we examined the effect of astaxanthin on TNFα- and palmitate-induced insulin resistance. The amount of reactive oxygen species generated by TNFα or palmitate with or without astaxanthin was evaluated by dichlorofluorescein staining. We also compared the effect of astaxanthin on insulin signaling with that of other antioxidants, α-lipoic acid and α-tocopherol. We observed astaxanthin enhanced insulin-stimulated GLUT4 translocation and glucose uptake, which was associated with an increase in insulin receptor substrate-1 tyrosine and Akt phosphorylation and a decrease in c-Jun N-terminal kinase (JNK) and insulin receptor substrate-1 serine 307 phosphorylation. Furthermore, astaxanthin restored TNFα- and palmitate-induced decreases in insulin-stimulated GLUT4 translocation or glucose uptake with a concomitant decrease in reactive oxygen species generation. α-Lipoic acid enhanced Akt phosphorylation and decreased ERK and JNK phosphorylation, whereas α-tocopherol enhanced ERK and JNK phosphorylation but had little effect on Akt phosphorylation. Collectively these findings indicate astaxanthin is a very effective antioxidant for ameliorating insulin resistance by protecting cells from oxidative stress generated by various stimuli including TNFα and palmitate.