Gastric cancer is the fourth most common malignant disease and second leading cause of cancer-associated mortalities worldwide. Previous studies revealed aberrantly expressed microRNAs (miRNAs) in various types of human cancer; these miRNAs play important roles in tumourigenesis and tumour development. miRNAs present a considerable potential for novel therapeutic approaches for treating human cancer. Therefore, the investigation of novel miRNAs involved in gastric cancer progression provides an opportunity to improve the prognosis of patients with gastric cancer. miRNA-28 (miR-28) has been investigated with regards to its expression and biological functions in many types of human cancer. However, previous studies have not discussed the expression patterns, roles and associated molecular mechanisms of miR-28 in gastric cancer. In the present study, miR-28 expression was identified to be upregulated in gastric cancer tissues and cell lines. miR-28 inhibition functionally inhibited cell proliferation and invasion in gastric cancer in vitro. Using bioinformatics analysis, luciferase reporter assay, reverse transcription-quantitative polymerase chain reaction and western blot analysis, phosphatase and tensin homolog (PTEN) was mechanically identified as a direct target of miR-28 in gastric cancer. PTEN was downregulated in gastric cancer and negatively correlated with miR-28 levels. Inhibition of PTEN restored the biological effects of miR-28 downregulation on the proliferation and invasion of gastric cancer cells. Notably, the downregulation of miR-28 results in the regulation of the phosphatidylinositol 3-kinase/protein kinase B signaling pathway in gastric cancer. These results suggested that miR-28 may be targeted for the development of novel treatments for gastric cancer in the future.