Background: Accumulating studies have shown that pseudogenes could become key regulators in human cancers. Misato family member 2, pseudogene (MSTO2P) is over-expressed in lung and gastric cancer and affects the biological functions of tumor cells. However, the role of MSTO2P in hepatocellular carcinoma (HCC) is unreported. Purpose: This study aimed to examine the diagnostic and prognostic value of MSTO2P in HCC, to investigate the effects of MSTO2P on the biological functions of HCC cells, and to explore the potential mechanisms of MSTO2P in HCC. Methods: Relevant data on HCC were downloaded from the Gene Expression Omnibus database and the Cancer Genome Atlas database and used to analyze MSTO2P expression and the role of MSTO2P in HCC prognosis. MSTO2P in HCC cell lines was knocked down by shRNA to study the effects of MSTO2P on cell proliferation, apoptosis, metastasis and invasion in HCC. Expressions of the main proteins involved in epithelial-mesenchymal transition and the PI3K/AKT/mTOR signaling pathway in HCC were examined via Western blot analysis. Results: MSTO2P had significant diagnostic and prognostic value in HCC. MSTO2P was highly expressed in HCC tissues and cells, and MSTO2P increased HCC cell proliferation, invasion and metastasis. MSTO2P knockdown also increased E-cadherin expression and decreased N-cadherin and Vimentin expression. Additionally, MSTO2P increased the expressions of proteins in the PI3K/AKT/mTOR pathway, including PI3K, p-AKT and p-mTOR. Conclusion: MSTO2P might be used as a potential target for diagnosing and curing HCC. MSTO2P may affect HCC cell proliferation, apoptosis, metastasis and invasion through the PI3K/AKT/mTOR pathway.