To evaluate the ameliorating effects of hydrogen peroxide (H2O2, 200 μM) on hexavalent chromium [Cr(VI)] toxicity in canola (Brassica napus L.), we focused on the plant growth, chlorophyll content, thiol contents, lipid peroxidation, antioxidant enzymes, and the expression of metallothionein protein (BnMP1) mRNA. Cr(VI) at 50 μM significantly decreased the plant growth (fresh and dry weights). The decrease in growth was accompanied by increased lipid peroxidation and decreased chlorophyll content in leaves. Hydrogen peroxide pretreatment, however, enhanced plant growth parameters and led to the reduced levels of lipid peroxidation and higher levels of pigment. In addition, H2O2 pretreatment increased Cr accumulation in aerial parts of seedlings. The tendency of increase in thiol content under Cr(VI) stress was further increased with H2O2 pretreatment. The activities of antioxidant enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidase (POD) and catalase (CAT) were differentially altered. SOD and POD activities increased under Cr(VI) stress, whereas APX and CAT activities decreased. The SOD and CAT activities remained unaffected in both durations due to H2O2 pretreatment, but activities of APX and POD were promoted in the Cr(VI)-stressed seedlings. Metallothioneins are a family of low-molecular-weight Cys-rich proteins and are thought to play a possible role in metal metabolism or detoxification. In real-time quantitative PCR analysis, the expression level of BnMP1 mRNA was increased at 1 day after treatment (DAT), whereas it was decreased at 7 DAT in Cr(VI)-stressed seedlings. At 1 DAT, pretreatment of H2O2 before Cr(VI) stress reduced the expression of BnMP1 mRNA as compared to Cr(VI) stress alone, but this effect was not significant. At 7 DAT, H2O2 pretreatment alleviated the Cr(VI) stress-mediated decrease in the expression of BnMP1 mRNA. These results suggest that H2O2 may act as a signal that triggers defense mechanisms which in turn protects canola seedlings from Cr(VI)-induced oxidative damage.