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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. Tryptophan decarboxylase (TDC) serves as the initial rate-limiting enzyme in the melatonin synthesis pathway. To date, no cucumber <i>TDC</i> gene has been cloned and characterized. In this study, we identified two <i>TDC</i> genes (<i>CsTDC1</i> and <i>CsTDC2</i>) in the cucumber genome. The subcellular localization analysis indicated that CsTDC1 and CsTDC2 are predominantly localized in the cytoplasm and plasma membrane. Tissue-specific expression analyses revealed that <i>CsTDC1</i> and <i>CsTDC2</i> 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 <i>CsTDC</i> promoter regions. Furthermore, the expression of <i>CsTDC1</i> and <i>CsTDC2</i> was strongly induced by treatments with various abiotic stresses and exogenous hormones. The transient overexpression of <i>CsTDC1</i> and <i>CsTDC2</i> in tobacco leaves resulted in increases in the TDC activity and melatonin content, along with improved tolerance of tobacco leaves to salt, drought, and low-temperature stresses. Notably, the overexpression of <i>CsTDC2</i> had a more pronounced effect than the overexpression of <i>CsTDC1</i>. Accordingly, both <i>CsTDC</i> genes, but especially <i>CsTDC2</i>, may be important for regulating cucumber growth, development, and stress tolerance. The study findings provide a theoretical and experimental basis for future functional analyses of endogenous melatonin in cucumber.