Variegation is a frequently observed genetic phenomenon in landscaping. In this study, an ethyl methanesulfonate induced variegated leaf ( Csvl) mutant in cucumber ( Cucumis sativus L.) was identified. The Csvl mutant displayed green-yellow-white variegation phenotype throughout the whole growth cycle, while the leaf of wild type plants was normal green. The photosynthetic pigment contents and photosynthetic parameters of Csvl was significantly lower than wild type. The cytology observation results showed that the mesophyll cells of Csvl mutant contained defective chloroplasts. Genetic analysis indicated that variegated leaf phenotype was monogenic recessive inheritance. MutMap and genotyping results revealed that Csa6G405290 ( Cscs), encoding chorismate synthase, was the candidate gene for variegated leaf mutant in cucumber. The expression level of Cscs was similar between wild type and variegated leaf mutant leaves. Transcriptome profile analysis of leaves of Csvl mutant identified 183 candidate genes involved in variegated leaf development in cucumber, including genes that encode heat shock protein, zinc finger protein. Cscs may regulate variegated leaf in cucumber by interacting with these genes. In a word, these results revealed that Cscs might regulate the variegated leaf phenotype in cucumber.