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Increases in global temperature and drought are negatively impacting the yields of major crops. Therefore, targeted improvements to intrinsic water use efficiency ( WUE _i ) are needed to reduce the water required for agricultural production. While it is very time-consuming to directly measure WUE _i , stable carbon isotope ratios (δ ¹³ C) are a reliable high throughput proxy trait for quantifying WUE _i in C ₃ species. While genetic studies have improved our understanding of the relationship between WUE _i and δ ¹³ C in C ₄ species, the knowledge needed to implement δ ¹³ C in breeding schemes is incomplete. Using a Zea mays line with an extremely negative δ ¹³ C value, a quantitative genetics approach was used to identify a large deletion in carbonic anhydrase1 ( cah1 ). Carbonic anhydrase is the first enzymatic step of the C ₄ photosynthetic pathway and is known to affect δ ¹³ C. Surprisingly, the line with the mutant allele has significantly higher carbonic anhydrase activity with a concurrent reduction in δ ¹³ C, opposite of what would be expected based on C ₄ carbon isotope fractionation theory. These observed decouple δ ¹³ C and WUE _i , which calls for further investigation into carbon isotope discrimination in C ₄ species.