Cadmium (Cd) is a toxic metal for living organisms. Humans are exposed to Cd mainly from the consumption of foods, among which vegetables are an important dietary source. Brassicas are important vegetables, but can also accumulate relatively high levels of Cd in the edible parts. It is therefore important to understand the mechanisms controlling Cd accumulation in Brassica vegetables. In a hydroponic experiment, we found large variations in the Cd concentrations in the roots and shoots, as well as the ratio of shoot to root Cd concentrations, among 64 cultivars of Brassica rapa. Because the P_1B-type heavy metal ATPase 3 (HMA3) has been shown to play an important role in controlling the root-to-shoot translocation of Cd in rice and Arabidopsis thaliana, we sequenced the BrHMA3 genes in all cultivars and found nine different alleles in the coding region, with five full-length alleles and four truncated alleles. Cultivars with the truncated alleles had on average x times higher Cd concentration in the shoots, and x times higher shoot to root Cd concentration ratio, than those possessing the full-length alleles. Heterologous expression of the nine BrHMA3 alleles in yeast showed Cd transport activities for the full-length alleles, but not for the truncated alleles, suggesting that the two types of alleles represent functional and weak or non-functional alleles, respectively. These two types of alleles explained 87% of the variance in the root-to-shoot Cd translocation among the 64 cultivars. We developed an F2 segregation population from a cross between a low-Cd cultivar with a functional allele and a high-Cd cultivar with a non-functional allele of BrHMA3. The BrHMA3 genotype explained 84.7% of the variance in the shoot to root Cd concentration ratio among the F2 progeny. Subcellular localization of BrHMA3 showed that it is localized at the tonoplast. Expression of a functional allele of BrHMA3 in both Arabidopsis thaliana and rice decreased the root-to-shoot translocation of Cd significantly. Taken together, our results show that allelic variation in the coding region of BrHMA3 is a key determinant of Cd accumulation in the shoots of Brassica rapa. Functional alleles of BrHMA3 can be used to breed low Cd cultivars.