Tomato is an important horticultural and economic crop cultivated worldwide. As Phytophthora infestans becomes a huge threat to tomato production, it is necessary to study the resistance mechanisms of tomato against P. infestans. Our previous research has found that miR482 might be involved in tomato– P. infestans interaction. In this study, miR482b precursor was cloned from Solanum pimpinellifolium “L3708” and miR482b was shown to decrease in abundance in tomato following P. infestans infection. Compared to wild-type tomato plants, tomato plants that overexpressed miR482b displayed more serious disease symptoms after P. infestans infection, with more necrotic cells, longer lesion diameters, and increased P. infestans abundance. Meanwhile, silencing of miR482b was performed by short tandem target mimic (STTM), resulting in enhancement of tomato resistance to P. infestans. Using miRNA and degradome data sets, NBS–LRR disease-resistance genes targeted by miR482b were validated. Negative correlation between the expression of miR482b and its target genes was found in all miR482b-overexpressing and -silencing tomato plants. Our results provide insight into tomato miR482b involved in the response to P. infestans infection, and demonstrate that miR482b– NBS–LRR is an important component in the network of tomato– P. infestans interaction.