Increases in global temperature and drought are negatively impacting the yields of major crops. Therefore, targeted improvements to intrinsic water use efficiency (WUEi) are needed to reduce the water required for agricultural production. While it is very time-consuming to directly measureWUEi,stable carbon isotope ratios (δ13C) are a reliable high throughput proxy trait for quantifyingWUEiin C3species. While genetic studies have improved our understanding of the relationship betweenWUEiand δ13C in C4species, the knowledge needed to implement δ13C in breeding schemes is incomplete. Using aZea maysline with an extremely negative δ13C value, a quantitative genetics approach was used to identify a large deletion incarbonic anhydrase1(cah1). Carbonic anhydrase is the first enzymatic step of the C4photosynthetic pathway and is known to affect δ13C. Surprisingly, the line with the mutant allele has significantly higher carbonic anhydrase activity with a concurrent reduction in δ13C, opposite of what would be expected based on C4carbon isotope fractionation theory. These observed decouple δ13C andWUEi, which calls for further investigation into carbon isotope discrimination in C4species.