Serotonin N-acetyltransferase (SNAT) and N-acetylserotonin methyltransferase (ASMT) are the last two key enzymes for melatonin biosynthesis in living organisms. In this study, we demonstrated that transgenic rice (Oryza sativa L.) plants, in which expression of either endogenous SNAT or ASMT was suppressed, had reduced melatonin synthesis, confirming that both SNAT and ASMT are functionally involved in melatonin synthesis. The melatonin-deficient SNAT rice had retarded seedling growth, which was partially restored by exogenous melatonin application, suggesting melatonin's role in seedling growth. In addition, the plants were more sensitive to various abiotic stresses, including salt and cold, compared with the wild type. Melatonin-deficient SNAT rice had increased coleoptile growth under anoxic conditions, indicating that melatonin also inversely regulates plant growth under anaerobic conditions with the concomitant high expression of alcohol dehydrogenase genes. Similarly, the melatonin-deficient ASMT rice exhibited accelerated senescence in detached flag leaves, as well as significantly reduced yield. These loss-of-function studies on the melatonin biosynthetic genes confirmed most previous pharmacological reports that melatonin not only promotes plant growth but also mitigates various abiotic stresses.