Gluconic acid was proposed as a prebiotic for nonruminants; however, a ruminant application is undescribed. The first experiment was a 5 × 5 Latin square with five test doses of calcium gluconate (CaG) at 0, 5, 17, 32, and 46 g d^-1. Treatments were infused into the abomasum of lactating dairy cows for 28 d and milk was collected on days 26, 27, and 28. The second experiment was a crossover where cows (N = 30) were fed a total mixed ration (TMR) with (0.2% of dry matter) and without CaG for 28 d. Milk (days 26, 27, and 28) and blood (day 28) were collected from all cows and rumen fluid (day 28) from a subset of six rumen-cannulated cows. Infusion of CaG into the abomasum increased milk fat yield and decreased dry matter intake resulting in improved feed efficiency. In contrast, cows fed CaG in the TMR had decreased milk yield and milk protein and lactose yields compared with control. In the subset of rumen-cannulated cows, the proportion of butyric acid was decreased and acetic acid increased relative to total volatile fatty acid in cows fed CaG. These findings suggest that CaG utilization may be different in the rumen and lower gastrointestinal tract.