The plant growth-promoting fungus (PGPF), Penicillium simplicissimum GP17-2 (GP17-2), induces systemic resistance against Pseudomonas syringae pv. tomato DC3000 (Pst) in Arabidopsis thaliana. The molecular mechanisms underlying induced systemic resistance (ISR) by GP17-2 were investigated in the present study. Microscopic observations revealed that stomatal reopening by Pst was restricted by elicitation with the culture filtrate (CF) from GP17-2. A gene expression analysis of MYB44, which enhances abscisic acid signaling and consequently closes stomata, revealed that the gene was activated by CF. CF-elicited myb44 mutant plants failed to restrict stomatal reopening and showed lower resistance to Pst than wild-type plants. These results indicate that stomatal resistance by GP17-2 is mediated by the gene activation of MYB44. We herein revealed that the MYB44-mediated prevention of penetration through the stomata is one of the components responsible for GP17-2-elicited ISR.