An efficient scheme to synthesize novel ring-A fused heterocyclic derivatives of betulin was developed. The starting reaction of this synthesis was one-pot selective bacterial oxidation of betulin to betulone used as the key compound to synthesize the substituted azoles such as C(2)-C(3)-fused 1,2,3-triazoles, oxazoles and 1,2,4-triazine, as well as C(1)-C(2)-fused isoxazoles. The semi-synthetic compounds were screened for their cytotoxic activity against human cancer cell lines A549, HCT 116, HEp-2, MS and RD TE32 with use of the photometric MTT assays. Among the tested compounds, N-acetyltriazole of betulin (10) displayed impressive cytotoxic activity with IC50 2.3-7.5 μM against HCT 116, HEp-2, MS and RD TE32 cell lines as well as 3-methyl-4-oxido-1,2,4-triazine-derivative of betulonic acid (12) that was active against HCT 116 and HEp-2 cell lines with IC50 1.4 and 1.5 μM, respectively. Comparative experiments showed triazole (10) to have a lower cytotoxicity to normal epithelial cells, in comparison with compound (12). In accord with the in vivo acute toxicity test, the LD50 of triazole (10) exceeded 600 mg/kg. The ability of the most potent active triazole (10) to trigger apoptotic cell death was explored in the Annexin V-FITC test and by analyzing of caspase activity and morphological alterations in mitochondria and nuclei of HCT 116 cells.