Surface-localized pattern recognition receptors perceive pathogen-associated molecular patterns (PAMPs) to activate pattern-triggered immunity (PTI). Activation of mitogen-activated protein kinases (MAPKs) represents a major PTI response. Here, we report that Arabidopsis thaliana PIF3 negatively regulates plant defense gene expression and resistance to Pseudomonas syringae DC3000. PAMPs trigger phosphorylation of PIF3. Further study reveals that PIF3 interacts with and is phosphorylated by MPK3/6. By mass spectrometry and site-directed mutagenesis, we identified the corresponding phosphorylation sites which fit for SP motif. We further show that a phospho-mimicking PIF3 variant (PIF3 /pifq) conferred increased susceptibility to P. syringae DC3000 and caused lower levels of defense gene expression in plants. Together, this study reveals that PIF3 is phosphorylated by MPK3/6 and phosphorylation of the SP motif residues is required for its negative regulation on plant immunity.