Melatonin is a crucial regulator of plant growth and development as well as stress tolerance. However, we only have a limited understanding of the functions of endogenous melatonin. Tryp-tophan decarboxylase (TDC) serves as the initial rate-limiting enzyme in the melatonin synthesis pathway. Unfortunately, no cucumber TDC gene has been cloned and characterized. In this study, we identified two TDC genes (CsTDC1 and CsTDC2) in the cucumber genome. The subcellular localization analysis indicated CsTDC1 and CsTDC2 are predominantly localized in the cyto-plasm and plasma membrane. Tissue-specific expression analyses revealed that CsTDC1 and CsTDC2 are expressed in both vegetative and reproductive organs. Many cis-elements related to stress, hormone, and light responses as well as development were identified in the CsTDC pro-moter regions. Furthermore, the expression of CsTDC1 and CsTDC2 was strongly induced by treatments with various abiotic stresses and exogenous hormones. The transient overexpression of CsTDC1 and CsTDC2 in tobacco leaves resulted in increases in the TDC activity and melatonin content, which may be related to the enhanced ability of tobacco leaves to tolerate salt, drought, and low-temperature stresses. Notably, the overexpression of CsTDC2 had a more pronounced effect than the overexpression of CsTDC1. Accordingly, both CsTDC genes, but especially CsTDC2, may be important for regulating cucumber growth, development, and stress tolerance. The study findings provide a theoretical basis for future functional analyses of endogenous melatonin in cucumber.