To further study the biotransformation process and producing enzyme properties of bacterial strain of Rhodobacter sphaeroides to HMX, the effects of different carbon sources, nitrogen sources and metal ions on the biotransformation efficiency of HMX of the strain and its growth were studied. The intermediate metabolites of HMX degraded by Rhodobacter sphaeroides were analyzed by liquid chromatography-mass spectrometry (LC-MS), and the possible degradation pathways were presumed. The effects of different conditions on the specific activity of the enzymes produced by the strain were determined. The zymograms were analyzed by polyacrylamide gel electrophoresis. The results show that the optimum carbon source, combined nitrogen source and metal ion for transforming HMX by the strain are malic acid, (NH₄)₂SO₄ and yeast extract, Ca²⁺, respectively. When the initial concentration of HMX is 100 mg·L^-1, after 96 h of culture, three substances can be detected: two HMX nitroso derivatives (mononitroso mNs-HMX and dinitroso dNs-HMX), methine dinitramine (MEDINA), and the mass-to-charge ratios of their mother ions are 279, 263 and 136, respectively. The possible degradation pathways presumed have two branches. One branche is that HMX is reduced to mNs-HMX and dNs-HMX by reductase, the other branche is that HMX is transformed and open-loop cleavaged into methine dinitramine by hydrolase. The specific activity of enzyme and polyacrylamide gel electrophoresis experiments show that the specific activity of the enzyme produced by the strain is significantly promoted when the concentration of HMX is 75 mg·L^-1 and 100 mg·L^-1, while when the concentration of HMX is 125 and 50 mg·L^-1, the specific activity of the enzyme produced by the strain has an inhibitory effect. When pH is 7, the specific activity of the enzyme produced by the strain is the highest.