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This study aims to reveal the role of nitric oxide (NO) in substituting nitrite with lactic acid bacteria. Three lactic acid bacterial strains (L. fermentum JCM1173, L. fermentum IFO3956, Weissella cibaria X31) with nitrite substitution ability were inoculated into fermented sausages, respectively. Results indicated that the ratio of nitrosomyoglobin (NO-Mb) to total pigments was positively correlated with the substitution ability of the strain. UV-visible and resonance Raman spectroscopy suggested the NO-Mb was five-coordinated. In addition, the intracellular NO detected by fluorescent DAF-FM probe showed that W. cibaria X31 had the strongest NO-producing ability. Inhibitory experiments showed that both nitrate reductase and nitric oxide synthase-like protein participated in the NO production. These results indicated the vital role NO played in the substitution through the formation of NO-Mb and demonstrated the sources of NO in lactic acid bacteria preliminarily.