Key message <p>A novel gene <i>CDE5</i> controlling leaf color was identified. Its causal gene <i>LOC_Os06g45980</i> was verified by complementation transgenic, expression analysis, RNA sequencing, enzyme activity tests.</p> Abstract <p>Leaf color is a critical trait influencing plant biosynthesis, as it directly impacts photosynthesis and dry matter production. Despite intensive research, the molecular mechanisms of leaf color still remain to be elucidated. In the present study, we characterized a rice <i>chlorophyll deficient 5</i> (<i>cde5</i>) mutant, which was defective in chloroplast development and exhibited albino leaves throughout the overall growth period. Map-based cloning revealed that <i>CDE5</i> encodes a DNA helicase which has not been previously reported to be associated with albino leaf in plants. The <i>cde5</i> mutant exhibited abnormal chloroplast structures and decreased chlorophyll contents. Further expression analyses showed that <i>CDE5</i> expressed in variety of tissues, and in situ hybridization results showed <i>CDE5</i> expressed in mesophyll cell. Subcellular localization analysis demonstrated that CDE5 was localized in the chloroplast and mitochondrion. RNA sequencing and RT-qPCR showed that several genes involved in chlorophyll synthesis, chloroplast development and photosynthesis were significantly altered due to loss of <i>CDE5</i>. Enzyme activity experiments further demonstrated that CDE5 exhibits ATPase activity, while the ATPase activity of the cde5 mutant protein is significantly reduced. Taken together, these findings suggest that <i>CDE5</i> plays an important role in leaf color regulation, thus shedding new light on the function of DNA helicase in rice.</p>