To analyze the function and regulatory mechanism of melatonin (N-acetyl-5-methoxytryptamine) in plant innate immunity, this study used the interaction between the pathogenic bacterium Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) and tobacco as a model system. The study examined the influence of pathogen invasion on the expression of tobacco genes related to melatonin and analyzed the effects and regulatory mechanisms of melatonin on pathogen grwoth, stomatal aperture, and ROS (reactive oxygen species) content in plant leaves. The results showed that: (1) Pst DC3000 treatment increased the expression of tobacco melatonin biosynthesis (NtSNAT1) and receptor (NtPMTR1) genes, and exogenous melatonin treatment reduced the bacterial contents in plant leaves. (2) Overexpression of the soybean GmSNAT1 significantly increased the endogenous melatonin concentrations and expression of NtPMTR1 in transgenic tobacco plants GmSNAT1-OE2 and GmSNAT1-OE5, and the Pst DC3000 bacteria growth number was significantly decreased. (3) Exogenous melatonin and bacterial flagellin peptide flg22 treatments induced the production of ROS and stomatal closure in both wild-type and transgenic tobacco guard cells, and the transgenic plants were more sensitive to melatonin- and flg22-induced stomatal closure and ROS production than the wild-type plants. Taken together, this study found that melatonin could reduce the bacteria invasion, which may probably be associated with promoting ROS production in guard cells and inducing stomatal closure via NtPMTR1-mediated signaling pathway.