Simple SummaryIn the past, zinc oxide was often used to control diarrhea in weaned piglets; however, it also resulted in a serious environmental burden. It is critical to identify safe and efficient substitutes for zinc oxide to prevent and treat diarrhea in weaned piglets. This study showed that tannic acid or antimicrobial peptides had better effects added to the feed as substitutes for zinc oxide to ensure the intestinal health of weaned piglets.AbstractZinc oxide (ZnO) harms the environment and can potentially increase the number of drug-resistant bacteria. Therefore, there is an urgent need to find safe and effective alternatives to improve gut health and reduce the incidence of diarrhea in weaned piglets. This study conducted an antibacterial test of ZnO, antibacterial peptides (AMPs), and tannic acid (TA) in vitro. Thirty piglets were randomly allotted to one of the following three dietary treatments: ZnO (2000 mg/kg ZnO diet), AMPs (700 mg/kg AMPs diet), and TA (1000 mg/kg TA diet). The results showed that the minimum inhibitory concentrations of ZnO and TA againstEscherichia coliandSalmonellawere lower than those of AMPs, and the minimum inhibitory concentrations of ZnO, AMPs, and TA againstStaphylococcus aureuswere the same. Compared to ZnO, AMPs increased the digestibility of dry, organic matter and the crude fat. Additionally, TA significantly (p< 0.05) increased the digestibility of dry and organic matter. On experimental day 14, the plasma interleukin-6 (IL-6) content of piglets supplemented with AMPs and TA was increased significantly (p< 0.05). On experimental day 28, alanine aminotransferase activity in the plasma of weaned piglets in the ZnO and TA groups was significantly (p< 0.05) higher than in piglets in the AMPs group. The levels of plasma IL-6 and immunoglobulin M (IgM) were significantly higher (p< 0.05) in the ZnO and AMPs groups than in the TA group. On experimental days 14 and 28, no significant differences were observed in the antioxidant capacity among the three experimental groups. Intestinal microbial diversity analysis showed that the Chao1 and ACE indices of piglets in the AMPs group were significantly higher (p< 0.05) than those in the ZnO and TA groups. At the genus level, the relative abundance ofTreponema_2was higher in the feces of piglets fed a diet supplemented with TA than in those fed diet supplemented with ZnO (p< 0.05). The relative abundance ofLachnospiraceaewas higher in the feces of piglets fed a diet supplemented with AMPs than in those fed diet supplemented with ZnO or TA. Overall, AMPs and TA could be added to feed as substitutes for ZnO to reduce diarrhea, improve nutrient digestibility and immunity, and increase the abundance of beneficial intestinal bacteria in weaned piglets.