The cytokine interleukin-6 (IL-6) mediates a wide range of inflammatory and immune responses. Tumor Necrosis Factor α (TNF-α) has a myriad of pro-inflammatory effects on the skin. In this study, we investigated the localizations and expression levels of IL-6, TNF-α and NF-κB in the skin of Rana dybowskii during the breeding period and pre-hibernation. Histologically, the skin of Rana dybowskii consists of epidermis and dermis. Four kinds of cells were identified in the epidermis during the breeding period and pre-hibernation, while the dermis was composed of homogenous gel, mucous glands and granular glands. IL-6, TNF-α and NF-κB were immunolocalized in the epithelial and glandular cells in both periods. Western blotting showed that IL-6, TNF-α and NF-κB were significantly higher in the pre-hibernation compared to the breeding period. Real-Time PCR revealed that the relative mRNA levels of IL-6 and NB-κB in the pre-hibernation increased significantly compared with the breeding period, while the TNF-α mRNA expression levels were no significant difference between these two periods. These results suggested that IL-6, TNF-α and NF-κB may collectively play important roles in the skin immune system of Rana dybowskii during the breeding period and pre-hibernation.