The present study unravels origin of nitric oxide (NO) and the interaction between 24-Epibrassinolide (EBL) and nitrate reductase (NR) for NO production in Indian mustard (Brassica juncea L.) under salinity stress. Two independent experiments were performed to check whether (i) Nitrate reductase or Nitric oxide synthase takes part in the biosynthesis of endogenous NO and (ii) EBL has any regulatory effect on NR-dependent NO biosynthesis in the alleviation of salinity stress. Results revealed that NR-inhibitor tungstate significantly (P ≤ 0.05) decreased the NR activity and endogenous NO content, while NOS inhibitor l-NAME did not influence NO biosynthesis and plant growth. Under salinity stress, inhibition in NR activity decreased the activities of antioxidant enzymes, increased H2O2, MDA, protein carbonyl content and caused DNA damage, implying that antioxidant defense might be related to NO signal. EBL supplementation enhanced the NR activity but did not influence NOS activity, suggesting that NR was involved in endogenous NO production. EBL supplementation alleviated the inhibitory effects of salinity stress and improved the plant growth by enhancing nutrients, photosynthetic pigments, compatible osmolytes, and performance of AsA-GSH cycle. It also decreased the superoxide ion accumulation, leaf epidermal damages, cell death, DNA damage, and ABA content. Comet assay revealed significant (P ≤ 0.05) enhancement in tail length and olive tail moment, while flow cytometry did not showed any significant (P ≤ 0.05) changes in genome size and ploidy level under salinity stress. Moreover, EBL supplementation increased the G6PDH activity and S-nitrosothiol content which further boosted the antioxidant responses under salinity stress. Taken together, these results suggested that NO production in mustard occurred in NR-dependent manner and EBL in association with endogenous NO activates the antioxidant system to counter salinity stress.