Cucumber is an agriculturally and economically important vegetable crop worldwide. Fruit flesh thickness is an important trait for cucumber and also a central determinant of yield, yet little is known about the underlying mechanism of this trait. In this study, bulked segregant analysis (BSA) combined with specific length amplified fragment sequencing (SLAF-seq) was applied to finely map the gene that underlies fruit flesh thickness in cucumber. A 0.19-Mb-long quantitative trait locus on chromosome 2 controlling fruit flesh thickness (QTL fft2.1) was identified and further confirmed by simple sequence repeat (SSR) marker-based classical QTL mapping in 138 F2 individuals. Gene prediction of this 0.19-Mb region identified 20 genes. Quantitative RT-PCR revealed higher expression levels of Csa2 M058670.1 (SET domain protein-lysine methyltransferase) in D8 (thick fruit flesh parent) compared with that in XUE1 (thin fruit flesh parent) during fruit development. Sequence alignment analysis of Csa2M058670.1 from thick and thin fruit flesh cucumber lines revealed a 4-bp deletion mutation in the promoter region of this candidate gene, which may result in the loss of Csa2M058670.1 activation in thin fruit flesh lines. The data presented herein suggest that Csa2M058670.1 is a possible candidate gene for controlling flesh thickness in cucumber.