Berberine (BBR) has previously been found to exert beneficial effects on renal injury in experimental rats. However, the mechanisms underlying these effects are not yet fully understood. Tumor necrosis factor (TNF) receptor-associated factor 5 (TRAF5) has been demonstrated to mediate the activation of nuclear factor-κB (NF-κB), which has been implicated in the pathogenesis of chronic kidney disease (CKD). The aim of this study was to investigate the effects of BBR on kidney injury and the activation of the NF-κB signaling pathway in mouse podocytes. TRAF5 was found to be overexpressed in patients with CKD and chronic renal failure (CRF) (data obtained from the dataset GSE48944, as well as from patients at Shuguang Hospital). TRAF5 overexpression significantly inhibited cell viability and induced the apoptosis of mouse podocytes. However, BBR prevented the decrease in cell viability and the apoptosis induced by TRAF5 overexpression. The NF-κB inhibitor, caffeic acid phenethyl ester (CAPE), mimicked the protective effects of BBR, as evidenced by the increased expression of nephrin and podocin, and the decreased the expression of caspase-3 and the ratio of Bax/Bcl-2. Moreover, BBR prevented the decrease in cell viability decrease and the apoptosis induced by TNF-α, interleukin (IL)-6 and lipopolysaccharide (LPS). Taken together, our data indicate that BBR exerts protective effects against CRF partly through the TRAF5-mediated activation of the NF-κB signaling pathway in mouse podocytes.