[Background] Microbial transformation is an important means for structural modification of natural products, featured with fast reaction, high selectivity, easy-controlled conditions and little pollution. Many microorganisms have been isolated for transformation of bufadienolides and several derivatives of reduced toxicity can be obtained by the modification at different positions. [Objective] The aim of this work is to find compounds with better bioactivity or reduced toxicity by screening the converting strains towards bufalin. [Methods] Substrate transformation experiments were carried out with bufalin as substrate to screen strains, and the products were identified by HPLC (High performance liquid chromatography) and LC-MS (Liquid chromatograph-mass spectrometer). The selected strain was determined in terms of colony morphology observation and molecular biological identification. The fermentation conditions were optimized to increase the conversion rate. The transformation of other steroids were also determined. [Results] One bufalin-transforming strain was obtained and identified as Naganishia sp. by the morphological characterisitics and evolutionary analysis on the basis of ITS sequence. The transformation product of this strain to bufalin was 3-ketobufalin. The optimal conversion conditions were determined with the initial pH value of 6.5, substrate concentration of 8 mg/L, the inoculation amount of 3% and the transformation time of 96 hours. About 48.3% of bufalin was converted into 3-ketobufalin. In addition, this strain could also transform the reversible reaction of estrone and 17β-estradiol. [Conclusion] It is first reported that Naganishia strain can transform bufalin into 3-ketobufalin, with less toxicity and great potential for safe drug to heart failure. The capability of transforming steroids of this strain also makes it possible to bioconvert and modify other steroidic compounds. The microbial conversion can provide a convenient path for large-scale production of the valuable compound because of high selectivity, mild reaction conditions and simple operation process.