Zeaxanthin (ZEA), a type of oxygenated carotenoid with strong antioxidant activity, has previously been found to exhibit an anti-lipogenesis effect. In the present study, we investigated the effect of ZEA on brown-like adipocyte formation and mitochondrial biogenesis in 3T3-L1 adipocytes. Brown adipocyte-specific markers, mitochondrial biogenesis and oxidative stress, and the involvement of AMP-activated protein kinase (AMPK) α1 were assessed. ZEA treated adipocytes demonstrated a brown-like pattern, with upregulated expression of uncoupling protein 1 (UCP1) and other brown adipocyte markers. In addition, ZEA intervention induced a dramatic increase in mitochondrial DNA (mtDNA) content and in the mRNA levels of genes associated with mitochondrial biogenesis. Furthermore, ZEA attenuated mitochondrial oxidative damage caused by lipid peroxidation in adipocytes, significantly improved the mitochondrial membrane potential (MMP), and scavenged intracellular reactive oxygen species (ROS) and mitochondrial superoxide. Finally, we concluded that AMPKα1 mediated the ZEA-caused inhibition of lipid accumulation and promotion of brown and beige adipocyte-biomarker expression, as the positive effects of ZEA were diminished by Prkaa1 (AMPKα1) knockdown. These findings demonstrated that ZEA promoted the expression of brown and beige adipogenesis markers and mitochondrial biogenesis, which involved AMPKα1 activation, thus contributing to the anti-obesity effects of ZEA.