Cucumber is one of the most widely consumed vegetables worldwide, and the fruit spine is an important fruit quality trait. Expansins play critical roles in fruit development; however, the regulation of expansins in cucumber fruit spine development has not been reported. In this study, 33 expansin genes were identified in the cucumber genome V3; additionally, expansin genes in Citrullus lanatus, Cucumis melo, Cucurbita maxima, Lagenaria siceraria, and Benincasa hispida were also identified. Phylogenetic analysis of expansin proteins in Cucurbitaceae and Arabidopsis showed that they evolved separately in each plant species. Phylogenetic analysis showed that C. maxima was derived earlier than the other five Cucurbitaceae species. The expression of CsEXPA2, CsEXPA14, and CsEXLA3 varied in cucumber lines with different fruit spine densities. A yeast two-hybrid assay showed that a putative auxin transporter encoded by numerous spine gene (ns) interacts with CsEXLA2, which may be involved in the development of the numerous spines in cucumber. These results provide novel insights into the expansins related to plant development and fruit spine development in cucumber.