N-acetylserotonin methyltransferase (ASMT) is among the key enzymes involved in the final steps of melatonin biosynthesis. Here, we have shown that inactivation of ASMT in A. thaliana results in reduced endogenous melatonin levels, modulating other plant hormone pathways and affecting stress-related responses. Transcriptomic analysis of the asmt-null mutant revealed that the differentially expressed genes were predominantly enriched in terms associated with auxin responses and signalling, as well as with abscisic acid (ABA)-mediated stress responses. In addition, the expression of genes involved in the ethylene, salicylic acid, jasmonic acid and brassinosteroid pathways was altered in the mutant. Assays of a β-glucuronidase (GUS) construct in which a fragment containing 1000 bp upstream of the ASMT start codon was fused to the GUS reporter gene confirmed that ASMT is involved in the responses to ABA, gibberellic and indole acetic acids, trans-zeatin, ethylene and epibrassinolide, which is consistent with the results of the in silico analysis of the ASMT promoter. Furthermore, the expression of a number of genes, such as SLG1, HIS1-3, AtAIRP1 and several LEA genes, whose transcriptional regulation is associated with water management and contributes to impaired tolerance to dehydration stress, was altered in the mutant. The pleiotropic effects of ASMT gene disruption facilitate the identification of new potential melatonin targets and provide insights into the specific mechanisms of melatonin action.