Ras homolog enriched in brain (Rheb) is a key regulator of mammalian target of rapamycin complex 1 (mTORC1). The Rheb-mTORC1 axis is a pivotal pathway that mediates cell growth. It was previously reported that upon energy-stress stimulation, the phosphorylation of Rheb at serine 130 by p38-regulated/activated protein kinase (PRAK) results in the impaired nucleotide binding ability of Rheb and inhibits Rheb-mediated mTORC1 activation. However, the role of Rheb phosphorylation in cancer development remains to be elucidated. The aim of the present study was to determine the effect of Rheb phosphorylation on tumor growth in vitro and in vivo. In addition, tissue samples were obtained from 70 hepatocellular carcinoma (HCC) patients in order to determine any associations between Rheb phosphorylation and the clinicopathological characteristics of patients. In vitro and ex vivo kinase assays were performed to determine the phosphorylation of Rheb by PRAK. A xenograft assay was performed to assess tumorigenicity of MEF cell lines. In addition, western blot and immunohistochemical analyses were performed to detect Rheb protein expression and phosphorylation. The results of the present study revealed that Rheb phosphorylation may be induced through Ras overexpression. In addition, kinase-dead PRAK and dominant-negative PRAK mutation were demonstrated to abolish the Rheb phosphorylation induced by Ras overexpression. Xenograft assays in nude mice revealed that Rheb phosphorylation was involved in PRAK-mediated tumor suppression. Of note, the clinicopathological analysis of 70 HCC samples determined that Rheb phosphorylation was associated with poor proliferation and the progression of HCC. In conclusion, the results of the present study suggested that Rheb phosphorylation may have an important role as an intracellular barrier to cancer development.