Male and female poplars (Populus cathayana Rehd.) respond differently to environmental stresses. However, little is known about sex-dependent responses to chilling at the proteome level. To better understand these differences, a comparative proteomics investigation combined with a biochemical approach was used in the current study. Three-month-old poplar cuttings were treated at 25 or 4 °C for 14 days. Results revealed significant sexual differences in nitrogen metabolic enzymes and free amino acid components in response to chilling. The chilling-treated males showed higher activities of nitrate reductase and glutamine synthetase and higher contents of reduced glutathione, serine, arginine, leucine, glycine, proline and methionine than chilling-treated females. A total of 65 chilling-responsive spots were found, of which 48 showed significant sexual differences. These proteins are involved in photosynthesis, carbon and energy metabolism, metabolic processes of proteins, lipid metabolism, vitamin metabolism, stress defense, and gene expression regulation. The study shows that males have more effective metabolic processes and protective systems to chilling than females.