We have evaluated the contribution of nitric oxide (NO) in static magnetic field (SMF-200 mT for 1h) induced tolerance towards UV-B stress in soybean seedlings using various NO modulators like sodium nitroprusside (SNP), inhibitor of nitrate reductase (NR) sodium tungstate (ST), NO synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) and diphenylene iodonium (DPI) a NADPH oxidase inhibitor. The UV-B exposure significantly reduced germination, seedling growth together with activities of total amylase, NOS and NR in seedlings from un-primed seeds whereas SMF-primed seedlings showed significant enhancement in all these parameters along with higher level of NO/ROS. The supply of NO donor, SNP further improved all the seedlings parameters in un-primed and SMF-primed seeds after UV-B exposure. While ST, L-NAME and DPI significantly reduced the SMF-induced seedling performance after UV-B exposure. The gene expression study also showed significant up-regulation of α-amylase (GmAMY1, GmAMY2), nitric oxide synthase (GmNOS2) and nitrate reductase (GmNR2) encoding genes in UV-B exposed SMF-primed seedlings over un-primed seedlings. In particular, SNP+UV-B treatment enhanced the GmNOS2 expression in both unprimed (31.9-fold) and SMF-primed (93.2-fold) seedlings in comparison to their respective controls of CK+UV-B. In contrast, L-NAME+UV-B treatment reduced the SMF-induced GmNOS2 expression (4.8-fold) and NOS activity (76%). It confirmed that NO may be the key signaling molecule in SMF stimulated tolerance towards UV-B stress during early seedling growth and NOS may possibly be accountable for SMF-triggered NO production in soybean seedlings exposed to UV-B irradiations.