In this research, the nitrate reduction rate increased 2-3 fold in the presence of five different porphyrin compounds (0.25 mM), among which hemin expressed the best accelerating effectiveness. Therefore, hemin was used to explore the catalytic characteristics and mechanisms during denitrification. The relationship between hemin concentrations (C-hemin) and nitrate reduction rates (k) could be best described by the equation k = 8.7463 + 0.44528ln (C-hemin-0.00993) (R-2 = 0.9908). Furthermore, the activation energy decreased 87% compared to the hemin-free system. Two active centers of hemin, the Fe3+ atom and the porphyrin ligand, might be involved in catalyzing the denitrification process. Additionally, the accelerating site of hemin in the denitrification electron transfer chain was elucidated by different metabolic inhibitors. This study provides a better understanding of porphyrin compounds in bio-multistage redox reactions and is a promising strategy for its practice application.