In this paper, the microbial transformation of desacetylcinobufagin (1) by Fusarium avenaceum AS 3.4594 was investigated, and four metabolites were isolated and characterized as 3-keton-desacetylcinobufagin (2), 3-epi-desacetylcinobufagin (3), bufadienolide A (4), and 15β,16α-dihydroxyl-17βH-bufalin (5), respectively. Among them, 4 and 5 are new compounds. The cytotoxicities of transformed products (2-5) against Hela cells were also investigated.