Pseudomonas aeruginosa is a nosocomial pathogenic bacterium that causes a wide range of human infectious diseases. The type III secretion system (T3SS) serves as a key virulence determinant underlying the pathogenesis of this bacterium in acute infections. As the master transcriptional activator of T3SS, ExsA binds to target promoter regions and modulates the expression of all currently identified T3SS genes. In this study, we identified tyrosine phosphoprotein A (TypA) as a repressor that restricts expression of the T3SS in P. aeruginosa. TypA interacts with ExsA to block its binding to target promoters, thereby inhibiting T3SS expression. We show that the typA expression is induced in response to low calcium, low temperature, a biofilm lifestyle, and direct contact with host cells. Additionally, the absence of TypA caused a growth defect in P. aeruginosa at low temperatures. Collectively, these data confirm the significant role of TypA and reveal a novel molecular mechanism by which P. aeruginosa regulates T3SS.