Objective To study the alkaloid constituents of the fruits of Melodinus yunnanensis (Apocynaceae). Methods The chemical constituents from the fruits of M. yunnanensis were isolated and purified by using silica gel, ODS, Sephadex LH-20 column chromatographies, TLC and preparative HPLC. The chemical structures were identified based on their physicochemical properties and spectral analysis. Results Twenty one alkaloids were isolated and elucidated. Compound 1 is a new one named melloyunnanine D (1), Compounds 1-21 were elucidated as scandine (2), scandine N-oxide (3), 10-hydroxyscandine (4), tabersonine (5), 11-methoxytabersonine (6), 11-hydroxytabersonine (7), lochnericine (8), pachysiphine (9), 11-hydroxylochnericine (10), 19R-hydroxytabersonine (11), 19R-acetoxytabersonine (12), voaphylline (13), 19S-vindolinine (14), 19R-vindolinine (15), venalstonidine (16), 17α-hydroxyvenalstonine (17), 19R-methoxytubotaiwine (18), kopsiyunnanines F1 (19), stemmadenine-N-oxide (20), stemmadenine (21). Conclusion The alkaloids in M. yunnanensis fruit were systematically recognized. Then, 21 compounds were isolated from M. yunnanensis fruits, among which compounds 1-4 belonged to monoterpene quinoline type alkaloid, 5-19 belonged to Aspidosperma type alkaloid, and compounds 20-21 belonged to Strychnos type alkaloid. Compounds 3-4, 7-12, 14-21 were isolated from this plant for the first time.