Wilms' tumor is a type of kidney cancer that affects young children. Although a number of Wilms' tumor samples have been collected through international trials, the mechanisms underlying its progression remain challenging to determine. Extensive studies have identified somatic mutations at several loci in Wilms' tumorigenesis, including WT1, catenin, Wilms' tumor gene on the X chromosome (WTX) and TP53. Berberine is a benzylisoquinoline alkaloid extracted from numerous types of medicinal plants and has been extensively used as a Chinese traditional medicine. Recently, berberine has been demonstrated to possess antitumoral activities. AMP-activated protein kinase (AMPK) is suggested to be one of the various cellular targets of berberine, which regulates tumor progression and metastasis. However, the specific involvement of berberine‑induced AMPK activation and its effects on the proliferation potential of Wilms' tumor cells remains unknown. The present study investigated the berberine‑induced activation of AMPK and its effects on G401 Wilms' tumor cell proliferation. The results demonstrated that berberine inhibited growth and decreased the expression of cell‑cycle regulators in these cells. At the molecular level, berberine treatment led to a significant increase of WTX expression and G401 cells were protected against berberine‑induced growth inhibition by small interfering RNA against WTX. In conclusion, these results suggest a novel mechanism that may contribute to the antineoplastic effects of berberine which was also demonstrated by recent population studies; however, further studies are required to investigate the potential therapeutic use of berberine in patients with Wilms' tumor.