Rubia cordifolia L. belongs to Rubiaceae family. Its medicinal parts are mainly root and rhizome, which is included in the Chinese Pharmacopoeia (2020), has the effect of cooling blood and stopping bleeding, removing stasis and channeling menstruation. However, studies on the hemostatic effect of R. cordifolia mainly focus on extracts, and the hemostatic components are less studied. Therefore, chemical constituents and coagulant activity of R. cordifolia were investigated in this paper. Eleven compounds were isolated and identified from R. cordifolia, including 1-hydroxy-2-methyl-9,10-anthraquinone (1), 1,4-dihydroxy-2-methylanthraquinone (2), palmitic acid (3), tricosanoic acid (4), ω-hydroxypachybasin (5), ursolic acid (6), oleanolic acid (7), rubilactone (8), β-sitosterol (9), cordifoliol (10), 1,3-dihydroxy-2-ethoxymethyl-9,10-anthraquinone (11). ω-Hydroxypachybasin was isolated from Rubia genus for the first time. 1,4-Dihydroxy-2-methylanthraquinone, rubilactone and 1,3-dihydroxy-2-ethoxymethyl-9,10-anthraquinone could remarkably shorten the plasma calcium coating time in vitro, thus indicating that they had procoagulant activity.