Acid rain is a widespread environmental issue intensely affecting normal plant growth of crops. Melatonin is well known pleiotropic molecule which improves abiotic and biotic stress tolerance of plants through physiological and molecular mediation. However, the impact of exogenous melatonin on molecular activities under acid rain conditions in plants has never been studied. The objective of the study is to expose the possible role of exogenous melatonin on physiological and molecular changes against acid rain stress in tomato. Transcriptome profile through RNA-sequence analysis identified 1228, 1120 and 1537 differentially expressed genes (DEGs) in control plant (Ctr) vs simulated acid rain stressed plant (P25) comparison, control plant vs melatonin treatment in simulated acid rain stressed plant (P25M) comparison and P25 vs P25M comparison, respectively. Among them, 152 differentially expressed genes (DEGs) were commonly expressed and the expression of secondary metabolites related gene was noticeably observed in all comparison. Moreover, transcript families such as ERF, WRKY, MYB and bZIP related gene accounted more in all treatment comparison. The RNA-sequence and qPCR results indicated that exogenous melatonin is closely associated with acid rain stress moderator and might be involved in alteration of differentially expressed genes (DEGs), biosynthesis of plant secondary metabolites and transcriptional factor encoding genes expression which might have potential application against environmental hazardous conditions. Display omitted •Acid rain causes oxidative damage and hampers growth, physiological and molecular activities in tomato.•Exogenous melatonin improved stress tolerance through stimulating the activities of antioxidants and directly ROS-scavenging free radicals.•Exogenous melatonin enhances acid rain stress tolerance in plants through up-regulating the secondary metabolites related gene expression.•Exogenous melatonin adjusts the expression of transcriptional factors to facilitate expression of ROS-scavenging enzyme-encoding genes.