Plant U-box (PUB) E3 ubiquitin ligases have undergone significant expansion compared to their fungal and animal counterparts. These E3 ligases play critical roles in diverse biological processes, including responses to biotic and abiotic stresses. However, systematic identification of PUB genes in cucumber (Cucumis sativus L.) has been lacking, and their expression and functional characterization remain largely unexplored. Leveraging the recently released near-complete cucumber genome, we identified 53 putative PUB proteins classified into eight distinct groups based on domain architecture. The molecular weights of CsPUBs range from 26 to 166 kilodaltons (kDa). Exon numbers in CsPUB genes vary substantially, with CsPUB48 containing a maximum of 17 exons, while 18 CsPUB genes harbor only a single exon. Chromosomal distribution of CsPUBs is uneven, with Chr 3 harboring the highest density (12 genes) and Chr 7 the lowest (1 gene). Notably, tandem duplications (e.g., CsPUB29-CsPUB36 and CsPUB18-CsPUB49) and seven collinear gene pairs were identified, suggesting evolutionary diversification. Promoter regions of CsPUBs are enriched with cis-regulatory elements linked to plant growth and development, phytohormone, stress responses, light, and so on, implying their regulatory roles in various biological processes. Expression profiling revealed tissue-specific patterns and differential regulation of multiple CsPUBs under stress conditions. Subcellular localization studies demonstrated that CsPUBs target diverse organelles, with some localizing to punctate structures potentially representing uncharacterized compartments. Collectively, this systematic analysis establishes a comprehensive framework for understanding particular CsPUB functions.