The pivotal role of ethylene (ETH) in fruit ripening has been extensively studied
however, the function of brassinosteroids (BRs) in regulating fruit ripening remains poorly understood. Specifically, the mechanism by which BRs interact with ETH to affect kiwifruit (Actinidia deliciosa) ripening is unclear. Our research showed that 2 genes encoding transcription factors, AdNAC3 and AdMYB19, and the fruit softening gene AdEXP3 (encoding a cell wall expansion protein, expansin 3) were upregulated by ETH and downregulated by BRs. Furthermore, AdNAC3 and AdMYB19 positively regulated the activity of the AdEXP3 promoter, and AdNAC3 positively regulated the promoter activity of AdMYB19. The physical interaction between AdNAC3 and the B-box-type zinc finger protein AdBBX32 affected fruit ripening. Transient overexpression and silencing experiments revealed that ETH upregulated and BRs downregulated the expression of AdNAC3 and AdMYB19, thereby regulating the expression level of AdEXP3 and participating in pectin degradation. Stable transformation of AdNAC3 in tomato fruits accelerated fruit color change and promoted fruit ripening. These results indicate that AdNAC3 and AdMYB19 are involved in the hormone interaction between BRs and ETH in regulating kiwifruit ripening, providing insights into the molecular mechanisms underlying the crosstalk between BRs and ETH.