Tomato is the highest-value fruit/vegetable crop worldwide. However, the quality and yield of tomatoes are severely affected by late blight. MicroRNA482s (miR482s) are involved in the plant's immune system. In this study, miR482c was transiently and stably overexpressed in tomatoes in transgenic plants to explore its mechanism in tomato resistance against late blight. Transgenic tomato plants with transiently overexpressed miR482c displayed a larger lesion area than the control plants upon infection. Furthermore, compared with wild-type (WT) tomato plants, the transgenic tomato plants stably overexpressing miR482c displayed a decreased expression of target genes accompanied by lower peroxidase (POD), superoxide dismutase (SOD), and phenylalanine ammonia-lyase (PAL) activity activities and higher malondialdehyde (MDA) content, thereby leading to a decline in reactive oxygen species (ROS) scavenging ability and aggravating the damage of lipid peroxidation product accumulation on the cell membrane, eventually enhancing plant susceptibility. This finding indicates that miR482c may act as a negative regulator in tomato resistance by regulating nucleotide binding sites and leucine-rich repeat (NBS-LRR) expression levels and ROS levels.