Three experiments (Exp) were conducted to characterize Zn appearance and disappearance in the rumen, rumen epithelial Zn importers and exporters, the directionality of Zn movement, and the effect of supplemental Zn on rumen epithelial structure. In Exp 1 and 2, ruminally-fistulated steers (n = 4) weighing 736 ± 23.3 kg were used in a crossover experiment with 2 dietary Zn concentrations [ZINC
0 (CON) or 120 mg supplemental Zn/kg DM (Zn120), from ZnSO4] in a 45% forage, 55% concentrate diet (Exp 1) or a 92.5% concentrate and 7.5% forage diet (Exp 2) to assess Zn appearance and disappearance in the rumen using the washed reticulo-rumen technique (WRR). Experiment 3 used twenty individually housed Hampshire crossbred wether lambs (35.1 ± 4.57 kg) fed for 60-d to evaluate the impact of dietary grain and Zn concentrations on ruminal epithelium Zn concentration, Zn transporters, and proteins integral to epithelial integrity. This Exp was a 2 × 2 factorial, with 2 diet types (DIET
45% forage and 55% concentrate [HF] or 7.5% forage and 92.5% concentrate [HG]), and 2 supplemental Zn treatments (ZINC
0 mg [CON] or 120 mg [ZN] supplemental Zn/kg diet DM) from ZnSO4. In Exp 1 and 2, ZINC did not affect the rate of disappearance (k) or plateau (B) digestive model parameters for buffer Zn (P ≥ 0.34) but ruminal Zn disappearance was numerically greater in Exp 2. In Exp 3, there was a DIET × ZINC effect (P = 0.05) where HG+ZN had the greatest rumen epithelial Zn, and HF+ZN had the least. There was an interaction between DIET and ZINC on papillae length (P = 0.05), where papillae length was shorter in HF than HG, and within HG, ZN animals had shorter papillae than CON (P = 0.01). A DIET × ZINC effect was noted for ZnT1 RNAscope score (P = 0.04) where HG+CON wethers had greater mean ZnT1 RNAscope score than other treatments (P ≤ 0.04). Ruminal Claudin-7 protein expression was lesser in HF (P = 0.01) and tended to be lesser in ZN (P = 0.09). Overall, these experiments highlight the dynamic nature of ruminal Zn metabolism and are supporting evidence for the relationship between ruminal health and dietary grain and Zn concentrations.