Fangchinoline (Fan) is a bioactive compound isolated from the Chinese herb S. Moore (Fen Fang Ji). The aim of the present study was to investigate the effect of Fan on the proliferation of SPC-A-1 lung cancer cells, and to define the associated molecular mechanisms. Following treatment with Fan, Cell Counting Kit-8, phase contrast imaging and Giemsa staining assays were used to detect cell viability; flow cytometry was performed to analyze the cell cycle distribution; and reverse transcription-quantitative polymerase chain reaction and western blot assays were used to investigate changes in the expression levels of cell cycle-associated genes and proteins. In the present study, treatment with Fan markedly inhibited the proliferation of SPC-A-1 lung cancer cells and significantly increased the percentage of cells in the G/G phase of the cell cycle in a dose-dependent manner (P<0.05 for 2.5-5 µm; P<0.01 for 10 µm), whereas the percentage of cells in the S and G/M phases were significantly reduced following treatment (P<0.05 for 5 µm; P<0.01 for 10 µm). Mechanistically, Fan significantly reduced the mRNA expression levels of cyclin D1, cyclin-dependent kinase 4 (CDK4) and CDK6 (P<0.05 for 2.5-5 µm; P<0.01 for 10 µm), which are key genes in the regulation of the G/G phase of the cell cycle. Furthermore, treatment with Fan also decreased the expression of phosphorylated retinoblastoma (Rb) and E2F transcription factor-1 (E2F-1) proteins (P<0.05 for 5 µm; P<0.01 for 10 µm). In summary, the present study demonstrated that Fan inhibited the proliferation of SPC-A-1 lung cancer cells and induced cell cycle arrest at the G/G phase. These effects may be mediated by the downregulation of cellular CDK4, CDK6 and cyclin D1 levels, thus leading to hypophosphorylation of Rb and subsequent suppression of E2F-1 activity. Therefore, the present results suggest that Fan may be a potential drug candidate for the prevention of lung cancer.