MADS-box proteins are important transcription factors that play essential roles in various aspects of plant development, particularly in flower development. In this study, we performed the identification and functional characterization of CsMADS09 isolated from cucumber (Cucumis sativus L.). CsMADS09 contains a 648-bp open reading frame encoding 215 amino acid residues, and shares high sequence identities with the members of the AP1/FUL family of MADS-box proteins, especially the euAPETALA1 (euAP1) subclade. Many cis-elements related to plant development, stress response, and hormones were identified in the promoter region of CsMADS09. Quantitative real-time polymerase chain reaction results showed that CsMADS09 was mainly expressed in reproductive tissues such as male flowers and unexpanded ovaries, while its expression was low in roots and only traceable in fertilized ovaries. Moreover, the results revealed that CsMADS09 expression tended to decline during male flower development and stayed nearly constant during female flower development. Ectopic expression of CsMADS09 resulted in earlier flowering and abnormal leaf development in transgenic Arabidopsis. This study is the first functional analysis of an AP1-like gene from cucumber and provides some clues for revealing the molecular mechanisms of flower development in cucumber.