BackgroundMagnesium chelatase plays an important role in photosynthesis, but only a few subunits have been functionally characterized in cassava.ResultsHerein,MeChlDwas successfully cloned and characterized.MeChlDencodes a magnesium chelatase subunit D, which has ATPase and vWA conservative domains.MeChlDwas highly expressed in the leaves. Subcellular localization suggested that MeChlD:GFP was a chloroplast-localized protein. Furthermore, the yeast two-hybrid system and BiFC analysis indicated that MeChlD interacts with MeChlM and MePrxQ, respectively. VIGS-induce silencing ofMeChlDresulted in significantly decreased chlorophyll content and reduction the expression of photosynthesis-related nuclear genes. Furthermore, the storage root numbers, fresh weight and the total starch content in cassava storage roots of VIGS-MeChlDplants was significantly reduced.ConclusionTaken together, MeChlD located at the chloroplast is not only required for chlorophyll biosynthesis and photosynthesis, but also affecting the starch accumulation in cassava. This study expands our understanding of the biological functions of ChlD proteins.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12870-023-04224-9.