The 14-3-3 proteins play essential roles in regulating various biological processes and abiotic stress responses in plants. However, there have been few studies of 14-3-3 family members in cucumber. Here, we identified a total of ten 14-3-3 genes (named as CsGF14a-j) in the cucumber genome. These genes are unevenly distributed across six cucumber chromosomes, and six of them were found to be segmentally duplicated. A phylogenetic analysis of 14-3-3 proteins in cucumber and other plant species showed that they could be divided into two distinct groups (ε and non-ε). Members in the same group tend to have similar exon-intron structure and conserved motif patterns. Several hormone-, stress- and development-related cis-elements associated with transcriptional regulation were found in the promoters of CsGF14 genes. RNA-seq data showed that most CsGF14 genes have broad expression in different tissues, and some had preferential expression in specific tissues and variable expression at certain developmental stages during fruit development. Quantitative real-time PCR (qRT-PCR) results revealed that nearly all tested CsGF14 genes were significantly up-regulated under cold and drought stress at certain time points. These results provide important information about the functions of CsGF14 genes in cucumber.