Pseudomonas aeruginosa (PA) expresses the type III secretion system (T3SS) and effector exoenzymes that interfere with intracellular pathways. Natural killer (NK) cells play a key role in antibacterial immunity and their activation is highly dependent on IL-12 produced by myeloid cells. We studied PA and NK cell interactions and the role of IL-12 using human peripheral blood mononuclear cells, sorted human NK cells, and a human NK cell line (NK92). We used a wild-type (WT) strain of PA (PAO1) or isogenic PA-deleted strains to delineate the role of T3SS and exoenzymes. Our hypotheses were tested in vivo in a PA-pneumonia mouse model. Human NK cells or NK92 cell line produced low levels of IFN-γ in response to PA without IL-12 stimulation, whereas PA significantly increased IFN-γ after IL-12 priming. The modulation of IFN-γ production by PA required bacteria-to-cell contact. Among T3SS effectors, exoenzyme T (ExoT) upregulates IFN-γ production and control ERK activation. In vivo, ExoT also increases IFN-γ levels and the percentage of IFN-γ[+] NK cells in lungs during PA pneumonia, confirming in vitro data. In conclusion, our results suggest that T3SS could modulate the production of IFN-γ by NK cells after PA infection through ERK activation.