Calcium ions are important second messengers, playing an important role in the signal transduction pathways. Hyperosmolality gated calcium-permeable channels (OSCA) gene family members play critical modulating roles in response to osmotic-related abiotic stress as well as other abiotic and biotic stresses, which has been reported in many plant species such as Arabidopsis, rice, maize, and wheat. However, there has been no report about the identification and expression profile of the OSCA genes in cucumber. In this study, a total of nine OSCA genes were identified, which are unevenly distributed on the six chromosomes of cucumber. Phylogenetic analysis revealed that the OSCAs of cucumber, Arabidopsis, rice and maize were clustered into four clades. The motif arrangement of CsOSCAs was strongly conserved, and the CsOSCA genes in each group had similar genetic structure. A total of 11 and 10 types of cis-elements related to hormone and stress, respectively, were identified in the promoter regions of CsOSCA genes. Gene expression analysis indicated that the CsOSCA genes have different expression patterns in various tissues, and some of them were regulated by three osmotic-related abiotic stresses (salt, drought and ABA) and three biotic stresses (powdery mildew, downy mildew, and root-knot nematode). As the first genome-wide identification and characterization of the OSCA gene family in cucumber, this study lays a foundation for research on the biological function and evolutionary process of this gene family, which is of great significance for exploiting stress resistant cucumber varieties.