Psa primarily utilises the type III secretion system (T3SS) to deliver effector proteins (T3Es) into host cells, thereby regulating host immune responses. However, the mechanism by which kiwifruit responds to T3SS remains unclear. To elucidate the molecular reaction of kiwifruit plants to Psa infection, M228 and mutant M228△hrcS strains were employed to inoculate Actinidia chinensis var. chinensis for performing comparative transcriptional and metabolomic analyses. Transcriptome analysis identified 973 differentially expressed genes (DEGs) related to flavonoid synthesis, pathogen interaction, and hormone signaling pathways during the critical period of Psa infection at 48 h post-inoculation. In the subsequent metabolomic analysis, flavonoid-related differential metabolites were significantly enriched after the loss of T3SS.Through multi-omics analysis, 22 differentially expressed genes related to flavonoid biosynthesis were identified. Finally, it was discovered that the transient overexpression of 3 genes significantly enhanced kiwifruit resistance to Psa. qRT-PCR analysis indicated that Ac4CL1, Ac4CL3 and AcHCT1 promote host resistance to disease, while Ac4CL3 negatively regulates host resistance to Psa. These findings enrich the plant immune regulation network involved in the interaction between kiwifruit and Psa, providing functional genes and directions with potential application for breeding kiwifruit resistance to canker disease.