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Dirigent proteins (DIRs) contribute to plant fitness by dynamically reorganizing the cell wall and/or by generating defense compounds during plant growth, development, and interactions with environmental stresses. ZmDRR206 is a maize DIR, it plays a role in maintaining cell wall integrity during seedling growth and defense response in maize, but its role in regulating maize kernel development is unclear. Association analysis of candidate genes indicated that the natural variations of <i>ZmDRR206</i> were significantly associated with maize hundred-kernel weight (HKW). <i>ZmDRR206</i> plays a dominant role in storage nutrient accumulation in endosperm during maize kernel development, <i>ZmDRR206</i> overexpression resulted in small and shrunken maize kernel with significantly reduced starch content and significantly decreased HKW. Cytological characterization of the developing maize kernels revealed that <i>ZmDRR206</i> overexpression induced dysfunctional basal endosperm transfer layer (BETL) cells, which were shorter with less wall ingrowth, and defense response was constitutively activated in developing maize kernel at 15 and 18 DAP by <i>ZmDRR206</i> overexpression. The BETL-development-related genes and auxin signal-related genes were down-regulated, while cell wall biogenesis-related genes were up-regulated in developing BETL of the <i>ZmDRR206</i>-overexpressing kernel. Moreover, the developing <i>ZmDRR206</i>-overexpressing kernel had significantly reduced contents of the cell wall components such as cellulose and acid soluble lignin. These results suggest that ZmDRR206 may play a regulatory role in coordinating cell development, storage nutrient metabolism, and stress responses during maize kernel development through its role in cell wall biogenesis and defense response, and provides new insights into understanding the mechanisms of kernel development in maize.