Late blight, caused by Phytophthora infestants (P. infestans), is severely damaging to the global tomato industry. MicroRNAs (miRNAs) have been widely demonstrated playing vital roles in plant resistance through repressing their target genes. Recently, clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein9 (CRISPR/Cas9), has been continuously improved and extensively applied to edit plants genomes. Though, editing multiplex miRNAs by CRISPR/Cas9 in tomato has not been studied yet. Herein, we knocked down miR482b and miR482c simultaneously in tomato through the multiplex CRISPR/Cas9 system. Two transgenic plants that silenced miR482b and miR482c simultaneously and one transgenic line silenced miR482b solely were obtained. Compared with wild type (WT) plants, the disease symptoms of three transgenic plants upon infection were alleviated accompanied by increased expression of their common target nucleotide-binding site-leucine-rich repeat (NBS-LRR) genes and declined levels of reactive oxygen species (ROS). Furthermore, silencing miR482b and miR482c simultaneously was more resistant than silencing miR482b solely in tomato. More importantly, we found that knocking down miR482b and miR482c can elicit expression perturbation of other miRNAs, hinting the cross regulation among miRNAs. Altogether, our study demonstrated that editing miR482b and miR482c simultaneously by CRISPR/Cas9 is an efficient strategy for generating pathogen-resistant tomatoes, and the cross regulation among miRNAs may reveal the novel mechanism in tomato- P. infestans interaction.