Abstract: In C₄ photosynthesis, pyruvate orthophosphate dikinase (PPDK) catalyzes the regeneration of phosphoenolpyruvate in the carbon shuttle pathway. Although the biochemical function of PPDK in maize is well characterized, a genetic analysis of PPDK has not been reported. In this study, we use the maize transposable elements Mutator and Ds to generate multiple mutant alleles of PPDK. Loss‐of‐function mutants are seedling lethal, even when plants were grown under 2% CO₂, and they show very low capacity for CO₂ assimilation, indicating C₄ photosynthesis is essential in maize. Using RNA‐seq and GC‐MS technologies, we examined the transcriptional and metabolic responses to a deficiency in PPDK activity. These results indicate loss of PPDK results in downregulation of gene expression of enzymes of the C₄ cycle, the Calvin cycle, and components of photochemistry. Furthermore, the loss of PPDK did not change Kranz anatomy, indicating that this metabolic defect in the C₄ cycle did not impinge on the morphological differentiation of C₄ characters. However, sugar metabolism and nitrogen utilization were altered in the mutants. An interaction between light intensity and genotype was also detected from transcriptome profiling, suggesting altered transcriptional and metabolic responses to environmental and endogenous signals in the PPDK mutants.