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.