The effects of Rhodiola extract (RE) on longevity and stress resistance in Caenorhabditis elegans (C. elegans), and the underlying molecular mechanisms were explored in the present study. Results showed that the lifespan of C. elegans was remarkably prolonged by 37.1% after treated with high-dose RE (480 μg mL-1). Intervention with RE alleviated aging-related declines in the C. elegans model, and enhanced the stress resistance against heat shock, ultraviolet radiation and paraquat. Moreover, RE reduced the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) and increased the activities of superoxide dismutase (SOD) and catalase (CAT). RE also upregulated the gene expression of sod-3, gst-4, daf-16, skn-1 in C. elegans, downregulated the gene expression of daf-2 and age-1, and accelerated the translocation of DAF-16 and SKN-1 into the nucleus. Furthermore, the daf-16(mu86) and skn-1(zu169) mutants reversed the extension of lifespan triggered by RE, indicating that these genes were involved in RE-regulated longevity. These results demonstrated that RE could enhance lifespan extension, healthspan and stress resistance of C. elegans via insulin/IGF signaling and SKN-1 pathways. Therefore, the present findings suggested Rhodiola as a potential candidate to ameliorate the symptoms of aging.