Both heat shock factors (Hsfs) and reactive oxygen species (ROS) are widely perceived to be involved in chilling stress response. However, the particular mechanism of Hsfs mediated-ROS metabolism related to chilling stress remains largely unknown, especially during chilling injury development of postharvest economically cold-sensitive fruit such as banana. In this study, we found that heat shock treatment (HST) could effectively alleviate postharvest banana chilling injury and reduce ROS accumulation during storage. The expression levels of several respiratory burst oxidase homolog (RBOH) genes, encoding ROS-producing enzymes, increased consistently with the progression, whereas were inhibited upon HST. Significantly, we identified an Hsfs MaHsf26, which exhibited a opposite expression pattern as that of MaRBOHs , as the putative binding protein of MaRBOHB-like1 , MaRBOHE-like and MaRBOHF-like promoters using yeast one-hybrid (Y1H) screening analysis. Further electrophoretic mobility shift assay (EMSA) and dual luciferase reporter (DLR) analysis displayed MaHsf26 suppressed the transcriptions of MaRBOHB-like1 , MaRBOHE-like and MaRBOHF-like through binding to the heat shock elements (HSE) in their promoters, respectively. In general, our results suggested that MaHsf26 plays as a transcriptional repressor of ROS generation by suppressing the transcriptions of MaRBOHs and thereby involving in the HST-mitigated chilling injury in postharvest banana fruit. • Heat shock treatment (HST) alleviated chilling injury (CI) of banana fruit. • Respiratory burst oxidase homolog (RBOH) gene expressions were inhibited by HST. • A nucleus-localized transcriptional repressor MaHsf26 was induced upon HST. • MaHsf26 directly suppressed the transcriptions of MaRBOHBs. [ABSTRACT FROM AUTHOR]