Abstract Puccinia striiformis f. sp. tritici (Pst) is an important obligate pathogen in wheat (Triticum aestivum L.) and secretes effectors into plant cells to promote infection. Identifying host targets of effector proteins and clarifying their roles in pathogen infection is essential for understanding pathogen virulence. In this study, we identified a serine-rich effector, Pst27791, from Pst that suppresses cell death in Nicotiana benthamiana. Stable overexpression of Pst27791 in wheat suppressed reactive oxygen species accumulation and the salicylic acid-dependent defense response. Transgenic wheat expressing the RNA interference construct of Pst27791 exhibited high resistance to Pst virulent isolate CYR31, indicating its importance in pathogenesis. Pst27791 interacting with wheat rapidly accelerated fibrosarcoma (Raf)-like kinase TaRaf46 in yeast and in planta. Knocking down TaRaf46 expression in wheat attenuated Pst infection and increased wheat immunity. The overexpression of TaRaf46 decreased wheat resistance to Pst and repressed MAPK activation in wheat. Pst27791 may stabilize TaRaf46 through the inhibition of proteasome-mediated degradation in N. benthamiana. The ability of Pst27791 to enhance Pst colonization was compromised when TaRaf46 was silenced, suggesting that the virulence of Pst27791 is mediated by TaRaf46. Overall, these results indicate that Raf-like kinase TaRaf46 is exploited by the Pst effector as a negative regulator of plant immunity to promote infection in wheat.