Extensive phytochemical study of the methanol extract of twigs and leaves of Buxus sinica resulted in the identification of forty-one Buxus alkaloids, including twenty undescribed ones, namely cyclobuxusinines A-I (1–7, 16 and 20), as well as secobuxusinines A-K (8–15 and 17–19). Their structures were delineated by detailed analysis using various spectroscopic techniques. cyclobuxusinines B (2) was the first Buxus alkaloid, whose CH3-18 was oxidized, implying the presence of special oxidative enzymes in this plant. Secobuxusinines C (10), D (11), and E (12), whose C-12 or C-19 have an OH group substitution, enriched the substituent pattern in Buxus alkaloid and suggested more structurally diverse alkaloids in the Buxus spp. In the assessment of their bioactivities, some of them exhibited significant cytotoxic effects on two human tumor ovarian cancer cell lines. Notably, compound 36 displayed more potent cytotoxic effect against ES2 and A2780 cell lines with the IC50 value of 1.33 μM and 0.48 μM, respectively. Forty-one metabolites, including twenty undescribed Buxus alkaloids, were isolated from the aerial part of Buxus sinica. Some of them showed good cytotoxic activity. Display omitted •1, Twenty undescribed and twenty-one known Buxus alkaloids were isolated from Buxus sinica.•2, Some Buxus alkaloids showed significant cytotoxicity against ES2 and A2780 cell lines.•3, Compound 36 displayed more potent cytotoxic with the IC50 value of 1.33 μM and 0.48 μM, respectively.