This study clarifies the mechanisms of Cd uptake, translocation and detoxification in Populus cathayana Rehder females and males, and reveals a novel strategy for dioecious plants to cope with Cd contamination. Females exhibited a high degree of Cd uptake and root-to-shoot translocation, while males showed extensive Cd accumulation in roots, elevated antioxidative capacity, and effective cellular and bark Cd sequestration. Our study also found that Cd is largely located in epidermal and cortical tissues of male roots and leaves, while in females, more Cd was present in vascular tissues of roots and leaves, as well as in leaf mesophyll. In addition, the distributions of sulphur (S) and phosphorus (P) were very similar as that of Cd in males, but the associations were weak in females. Scanning electron microscopy and energy spectroscopy analyses suggested that the amounts of tissue Cd were positively correlated with P and S amounts in males, but not in females (a weak correlation between S and Cd). Transcriptional data suggested that Cd stress promoted the upregulation of genes related to Cd uptake and translocation in females, and that of genes related to cell wall biosynthesis, metal tolerance and secondary metabolism in males. Our results indicated that coordinated physiological, microstructural and transcriptional responses to Cd stress endowed superior Cd tolerance in males compared with females, and provided new insights into mechanisms underlying sexually differential responses to Cd stress.