Abstract： Objective To prepare rat heme oxygenase-1 (HO-1) mutants and todetermine the activity and inhibition of this mutated enzyme. Methods pcDNA3HO1 containing truncated native rat HO-1 cDNA and pcDNA3HO1Δ25 carrying mutated rat HO-1 cDNA (His25Ala) were constructed, respectively. COS-1 cells transfected with pcDNA3HO1 and pcDNA3HO1Δ25 were collected and their activities were analyzed. Results Native rat HO-1 was highly expressed in transfected cells and its activity was 13?688-15?600 U/mg protein per hour. However, the enzyme activity of mutated HO-1 declined and the value was 1948-2160 U/mg protein per hour. When an equal amount of mutant was added to the enzyme reaction system, the level of bilirubin decreased by 42%. Conclusion The His25Ala mutant reduced the formation of bilirubin, suggesting that the mutant could competely bind the heme with native enzyme.