In this paper, microbial transformation of resibufogenin by Fusarium solani AS 3.1829 was investigated, and five transformed products were isolated and identified as 3-ketone-resibufogenin (2), 3-one-cyclic 3-(1,2-dimethyl-1,2-ethanediylacetal)-resibufogenin (3), 3-dimethoxyl-resibufogenin (4), 3-epi-resibufogenin (5), and 3-epi-15α-hydroxy-7βH-bufalin (6), respectively. Among them, 3, 4, and 6 are new compounds, and the rare double oxidization of C-3 was reported. In addition, the cytotoxicities of transformed products were also investigated.