Drought stress severely affects cotton productivity and seedling survival. Resurrection plants are known for their unique mechanisms of desiccation tolerance, including the maintenance of photosynthetic proteins during dehydration and rehydration, making their genes valuable for drought-tolerant cotton breeding. Chloroplast DnaJ proteins play roles in protein quality control in plant cells. Here, we report the identification and functional characterization of a chloroplast-localized C-type DnaJ protein-coding gene BhDnaJC6 from the resurrection plant Boea hygrometrica. BhDnaJC6 transcripts accumulate in response to slow desiccation, and rapid desiccation in acclimated (desiccation-tolerant) but not non-acclimated (desiccation-sensitive) B. hygrometrica plants. Microscopic observation confirmed the cellular localization of BhDnaJC6-GFP in chloroplasts in transiently transformed tobacco guard cells, and its interference with Rieske iron–sulfur protein, the PetC subunit of the cytochrome b6/f complex, fused with mCherry. In silico analysis predicted a possible physical interaction between BhDnaJC6 and Rieske iron–sulfur protein, which was experimentally confirmed using bimolecular fluorescence complementation (BiFC) and yeast two-hybrid assays. When overexpressed in cotton, the BhDnaJC6 transgenic lines displayed higher Rieske iron–sulfur protein levels and improved drought tolerance compared to the wild type. The higher levels of Rieske iron–sulfur protein improve photosynthetic performance in transgenic lines under both non-stressed and drought-stressed conditions, increasing the electron transport rates and actual quantum yields of PSII and decreasing the quantum yield of non-regulated energy dissipation. Taken together, our findings unveil a novel component enhancing Rieske iron–sulfur protein stability and improving the drought tolerance of transgenic cotton, offering a valuable genetic resource for drought-tolerant cotton breeding.