Sclerotinia sclerotiorum was one of the serious diseases in B. napus, which could result in large loss of the yield of B. napus. In previous study, we identified the candidate GST gene, BnGSTU12, associated with sclerotiorum stem resistance and the expression levels of BnGSTU12 in resistant lines were higher than that in susceptible lines. We analyzed the function of BnGSU12 in B. napus in this study. The expression level of BnGSTU12 increased significantly at 24 h after S. sclerotiorum infection. Transgenic functional analysis indicated that overexpression of BnGSTU12 in A. thaliana and B. napus and transient expression of BnGSTU12 in tobacco enhanced S. sclerotiorum resistance, whereas virus-induced gene silencing (VIGS)-mediated BnGSTU12 silencing reduced S. sclerotiorum resistance. The inoculated BnGSTU12-OE A. thaliana and B. napus plants showed higher antioxidant enzyme activity and lower H ₂O ₂ content than WT. BnGSTU12 was rapidly induced by the hormone SA and MeJA, and we investigated whether the JA and SA pathway was involved in GSTU12 mediated S. sclerotiorum resistance. JA content was higher in the inoculated BnGSTU12-OE than WT plants. In addition, the expression level of JAR gene involving in JA-Ile biosynthesis and JA-responsive genes ( PDF1.2) were higher, JAZ gene repressing JA signaling was less in OE plants than WT after 12 and 24 h inoculation with S. sclerotiorum. The results showed that BnGSTU12 enhanced S. sclerotiorum resistance through ROS homeostasis and JA signaling pathway.