Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium in the environment and a leading cause of nosocomial infections worldwide. Therefore, it is listed by the World Health Organization as one of the human pathogens that urgently need to develop new antibacterial drugs. Recent findings have demonstrated that eukaryote-type Ser/Thr protein kinases play a vital role in regulating various bacterial physiological processes by catalyzing protein phosphorylation. Stk1 has been proved to be a Ser/Thr protein kinase in P. aeruginosa. However, the regulatory roles of Stk1 have not yet been revealed. Thus, we constructed the stk1 knockout mutant (∆stk1) from the P. aeruginosa PAO1 strain and employed a TMT labeling-based quantitative proteomic strategy to characterize proteome-wide changes in response to stk1 knockout. In total, 620 differentially expressed proteins, among which 288 proteins were upregulated and 332 proteins were downregulated, were identified in the ∆stk1 compared with the P. aeruginosa PAO1. Detailed bioinformatics analysis of these differentially expressed proteins were performed, including GO annotation, protein domain profile, the KEGG pathway, subcellular localization as well as enrichment analysis. Notably, downregulation of type Ⅳ pilus-related proteins and upregulation of T6SS-H1-related proteins were found in ∆stk1 strain, and the results were corroborated by quantitative PCR at the mRNA level. Further experiments confirmed that the loss of stk1 weakens the bacterial twitching motility and promotes a growth competition advantage, which is mediated by type Ⅳ pilus related proteins and T6SS-H1 related proteins, respectively. These findings contribute to a better understand the physiological role of Stk1, and the proteomic data will help further investigations of the roles and mechanisms of Stk1 in P. aeruginosa, though the detailed regulation and mechanism of Stk1 need to be further revealed.