MicroRNA (miRNA) critically controls gene expression in many biological processes, including lung growth and pulmonary surfactant biosynthesis. The present study was conducted to investigate whether miR-20a-5p had such regulatory functions on alveolar type II (AT-II) cells. To accomplish this, miR-20a-5p-overexpressed and miR-20a-5p-inhibited adenoviral vectors were constructed and transfected into cultured AT-II cells that were isolated from rat foetal lungs of 19 days' gestation. Transfection efficiency was confirmed by observing the fluorescence of green fluorescent protein (GFP) carried by the viral vector, whereas miR-20a-5p levels were verified by real-time PCR. The CCK-8 assay was used to compare the proliferation ability of AT-II cells that had over- or underexpressed miR-20a-5p. The expression of surfactant-associated proteins (SPs) and phosphatase and tensin homolog (PTEN) was measured by real-time PCR and Western blotting. In AT-II cells, transfection resulted in over- or under-regulation of miR-20a-5p. While overexpression of miR-20a-5p promoted pulmonary surfactant gene expression, its underexpression inhibited it. Consistent with its role in negatively regulating the pulmonary surfactant gene, an opposite pattern was observed for miR-20a-5p regulation of PTEN. As a result, when miR-20a-5p was rendered overexpressed, PTEN was down-regulated. By contrast, when miR-20a-5p was underexpressed, PTEN was up-regulated. Neither overexpression nor underexpression of miR-20a-5p altered the cell proliferation. miR-20a-5p plays no role in proliferation of foetal AT-II cells but is a critical regulator of surfactant gene expression. The latter appears to be achieved through a regulatory process that implicates expression of PTEN.