Sclerotinia sclerotiorum, the causal agent of Sclerotinia stem rot (SSR) on more than 450 plant species, is a notorious fungal pathogen. Nitrate reductase (NR) is required for nitrate assimilation that mediates the reduction of nitrate to nitrite and is the major enzymatic source for NO production in fungi. To explore the possible effects of nitrate reductase SsNR on the development, stress response, and virulence ofS. sclerotiorum, RNA interference (RNAi) ofSsNRwas performed. The results showed thatSsNR-silenced mutants showed abnormity in mycelia growth, sclerotia formation, infection cushion formation, reduced virulence on rapeseed and soybean with decreased oxalic acid production. FurthermoreSsNR-silenced mutants are more sensitive to abiotic stresses such as Congo Red, SDS, H2O2, and NaCl. Importantly, the expression levels of pathogenicity-related genesSsGgt1,SsSac1, andSsSmk3are down-regulated inSsNR-silenced mutants, whileSsCypis up-regulated. In summary, phenotypic changes in the gene silenced mutants indicate that SsNR plays important roles in the mycelia growth, sclerotia development, stress response and fungal virulence ofS. sclerotiorum.