Attention is increasingly being paid to the contamination of soil by the radioactive element uranium (U). Phytoremediation of contaminated soil by economically advantageous and environment-protective plants shows considerable potential for addressing this problem. Populus cathayana is a species with high heavy-metal tolerance, economic value, and notable potential for phytoremediation. Plant-sex-related differences can lead to differences in vegetative growth and tolerance to various stressors. As such, in this study, we designed a pot experiment to analyze the responses of male and female trees of P. cathayana to 50 mg kg−1 U stress in contaminated soil for 3 months. We studied the U uptake and distribution, photosynthesis, chlorophyll fluorescence, active oxygen species, and antioxidant enzymes of P. cathayana. The results showed that the photosynthetic activity and chlorophyll fluorescence of male and female trees were similar, and U stress mainly affected the nonstomatal factors and photosystem II during photosynthesis. Regarding the physiological and biochemical processes, male and female trees showed different defense strategies: male trees had higher peroxidase (POD), H2O2, and soluble sugars, but lower malondialdehyde (MDA), superoxide dismutase (SOD), and soluble proteins. Under U stress, the active oxygen produced by male trees could be cleared by antioxidant enzymes, preventing damage to the cell membrane. Male trees accumulated a higher U concentration in their roots than female trees, whereas the transportation of U from roots to leaves in male trees was lower than that in female trees. Therefore, our results suggested that male trees have a higher tolerance capacity and greater ability to remediate U-polluted soil than female trees. Future phytoremediation studies should consider the differences between plant sexes in the tolerance to U-contaminated land.