The aim of the present study was to evaluate the effects of baicalein on human endometrial stromal cells in vitro. Ectopic endometrium samples were obtained from 6 female patients with ovarian endometriosis who underwent laparoscopic surgical procedures from July to September 2015. After culturing the cells, immunocytochemistry was performed to verify the purity and homogeneity of the endometrial stromal cells, and a Cell Counting Kit-8 assay was used to evaluate cell viability. In addition, cell cycle progression was analyzed using flow cytometry, and the effects of baicalein on the expression of B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), proliferating cell nuclear antigen (PCNA) and cyclin D1 in endometrial stromal cells were evaluated using western blot analysis. The related signaling pathways were also investigated by incubating cells with inhibitors of signaling pathways, prior to adding 40 µM baicalein for 48 h, followed by analysis of cell viability using a Cell Counting Kit-8 assay. The results indicated that treatment with baicalein significantly induced a dose-dependent decrease (P<0.05) in the viability of human endometrial stromal cells, which was abolished by inhibition of the nuclear factor (NF)-κB signaling pathway. However, baicalein treatment did not induce a time-dependent decrease in viability, as cell viabilities between the 24, 48 and 72 h treatment groups did not differ significantly. The number of cells in the G0/G1 phase significantly increased following treatment with baicalein (P<0.05), while the number of cells in the S and G2/M phases significantly decreased (P<0.05). Baicalein-treated cells also exhibited significantly reduced expression of Bcl-2, PCNA and cyclin D1 compared with control cells (P<0.05). These results suggested that baicalein may suppress the viability of human endometrial stromal cells through the NF-κB signaling pathway in vitro, and may induce apoptosis and promote cell cycle arrest at the G0/G1 phase. Thus, baicalein may provide a novel treatment option for endometriosis.