SpWRKY3 was identified as a resistance gene to Phytophthora infestans from Solanum pimpinellifolium L3708 and its transgenic tomato showed a significant resistance to P. infestans. This finding reveals the potential application of SpWRKY3 in future molecular breeding. Transcription factors (TFs) play crucial roles in the plant response to various pathogens. In this present study, we used comparative transcriptome analysis of tomatoes inoculated with and without Phytophthora infestans to identify 1103 differentially expressed genes. Seven enrichment GO terms (level 4) associated with the plant resistance to pathogens were identified. It was found that thirty-five selected TF genes from GO enriched term, sequence-specific DNA binding transcription factor activity (GO: 0003700), were induced by P. infestans. Of these TFs, the accumulation of a homologous gene of WRKY (SpWRKY3) was significantly changed after P. infestans induction, and it was also isolated form P. infestans-resistant tomato, Solanum pimpinellifolium L3708. Overexpression of SpWRKY3 in tomato positively modulated P. infestans defense response as shown by decreased number of necrotic cells, lesion sizes and disease index, while the resistance was impaired after SpWRKY3 silencing. After P. infestans infection, the expression levels of PR genes in transgenic tomato plants overexpressed SpWRKY3 were significantly higher than those in WT, while the number of necrotic cells and the reactive oxygen species (ROS) accumulation were fewer and lower. These results suggest that SpWRKY3 induces PR gene expression and reduces the ROS accumulation to protect against cell membrane injury, leading to enhanced resistance to P. infestans. Our results provide insight into SpWRKY3 as a positive regulator involved in tomato-P. infestans interaction, and its function may enhance tomato resistance to P. infestans.