Tonoplast sugar transporters (TSTs) play essential roles in regulating plant growth, development, and response to various biotic and abiotic stresses. In this study, a total of three TST genes were identified by a genome-wide analysis in cucumber. Phylogenetic analysis showed that TST proteins from cucumber and other plant species can be classified into five groups, and nearly all TST members in the same groups displayed similar motif distributions, transmembrane (TM) domains, and gene structures. All of the three CsTST genes possess a number of development-, stress-, and hormone-related cis-elements in the promoter sequences. Meanwhile, qRT-PCR assays revealed that the CsTST1 was expressed in fruits, flowers, leaves, and other tissues, and its expression varied significantly under various abiotic stresses such as cold, salt, drought (PEG), and abscisic acid (ABA). Finally, functional analysis of CsTST1 in yeast revealed that it was able to complement the deficiency in galactose, mannose and sucrose transport. These results revealed that CsTST1 can act as a functional sugar transporter to play important roles in cucumber growth and response to abiotic stress probably through affecting carbohydrate distribution.