This study is to explore the active ingredients of traditional Chinese medicine rhubarb with antiproliferative activity on hypertrophic scar fibroblasts (HSF). Rhubarb was extracted with Soxhlet extraction method by different polar solvents. MTS method was used to screen rhubarb solvent extracts (25 microg x mL(-1)) with anti-proliferative activity on HSF, and flow cytometry was used to detect their influences on cell cycle. Then, the active ingredients were analyzed by HPLC. The components with high activity were identified by UPLC-Q/TOF and verified by HE staining. The results showed that the ethyl acetate extract of rhubarb had higher anti-proliferative activity (P < 0.01), increased significantly the proportion of cells in G0/G1 phase (P < 0.01), and reduced the proliferation index (PI) (P < 0.01). The main active ingredients were anthraquinones. The results of confirming experiment showed that emodin, rhein and gallic acid could inhibit cell proliferation in a dose-dependent manner. In conclusion, the ethyl acetate extract of rhubarb showed anti-proliferative activity on HSF, and the anti-proliferative ingredients might be anthraquinones.