Heme oxygenase-1 (HO-1) is a stress-responsive gene coding for an enzyme catalyzing the catabolism of heme to yield biliverdin IXα, carbon monoxide (CO) and iron. However, its biological role in regulating metal homeostasis, particularly the tolerance to toxic heavy metals is poorly understood. In this study, a novel gene encoding a Brassica juncea heme oxygenase-1 (designated as BjHO-1) was cloned and functionally identified. Spatial expression of BjHO-1 showed that it was differentially expressed in cotyledon, hypocotyl, leaf and root. BjHO-1 was found to be induced significantly by heavy metal Hg. To understand whether BjHO-1 is able to regulate plant tolerance to Hg, we constructed transgenic B. juncea plants overexpressing HO-1, and showed that 35S::BjHO1 plants confer the plant resistance to Hg toxicity by improving plant dry mass, reducing Hg accumulation, and attenuating Hg-induced oxidative stress. We further cloned a 1,099 bp promoter sequence upstream of BjHO-1 using genome walking approach. Multiple stress-responsive elements were detected in the BjHO-1 promoter regions. The promoter can be activated by Zn, Cd, Hg and Pb exposure. Our results indicate that up-regulation of BjHO-1 is beneficial for limiting the uptake or accumulation of heavy metals into plants. This work also provides a new example for molecular breeding designed for plants that do not accumulate or minimizing accumulation of toxic trace metals growing on heavy metal-contaminated soils.