Objective To explore the effect of sitostero-3-O-glucoside on proliferation, apoptosis and collagen synthesis of keloid fibroblasts (KF). Methods Cell viability was measured by MTT assay after cells were cultured with 0 (control), 3.125, 6.25, 12.5, 25, 50, 100 μg/mL sitostero-3-O-glucoside. Cell proliferation was measured by EdU staining. Cell apoptotic rate and cell cycle were measured by flow cytometry. The level of COL I and COL Ⅲ was detected by enzyme linked immunosorbent assay (ELISA). Results 3.125, 6.25, 12.5, 25, 50, 100 μg/mL sitostero-3-O-glucoside reduced cell viability (P< 0.01), increased inhibitory rate of cell proliferation (P< 0.01), IC₅₀=27.54±2.18 μg/mL. Compared with the control group, 12.5, 25, 50 μg/mL sitostero-3-O-glucoside reduced the cell proliferation rate (P< 0.01), increased the cell early and late apoptotic rates (P< 0.01), the cell cycle was arrested at G1 phase (P< 0.01), and the levels of COL I and COL Ⅲ were down-regulated (P< 0.01). Conclusions The results show that sitostero-3-O-glucoside can significantly inhibit the proliferation, induce cell apoptosis and inhibit the synthesis of collagen of keloid fibroblasts.