Banana fruit is prone to chilling injury (CI) during cold storage, resulting in quality deterioration and commodity reduction. The hot water treatment (HWT), dipping banana fruit in hot water (52 °C) for 3 min, reduced CI symptom at 7 °C storage. The purpose of this study was to investigate the potential molecular mechanism of HWT on the alleviation of CI of postharvest banana fruit. It was found that HWT treatment obviously inhibited the increases in CI index, relative electrolytic leakage, and the contents of malonaldehyde (MDA) and O₂^•-, while enhanced proline accumulation. Further transcriptome analysis in the pericarp of banana fruit was evaluated during storage. The results showed that differentially expressed genes (DEGs) in the comparison between control and HWT group were mainly enriched in photosynthesis, chlorophyll metabolism, lipid metabolism, glutathione metabolism, and brassinosteroid and carotenoid biosynthesis. Moreover, transcriptome expression profiles and RT-qPCR analyses exhibited that the corresponding genes involved in these metabolism pathways and heat shock proteins (HSPs) were upregulated by HWT during cold storage. In general, our findings clearly reveal the potential pathways by which HWT alleviates CI in banana fruit, enriching the theoretical basis for the application of hot water to reduce CI in fruits.