Simple SummaryDiarrhea is the main cause of death in newborn calves and is associated with antibiotic use and economic loss for dairy farms. In this study, we evaluated the effects of different sources of the mineral zinc (zinc oxide (ZnO) and zinc methionine (Zn-Met)) on the growth, incidence of diarrhea, tissue zinc accumulation, gene expression of jejunal zinc transporters and serum concentrations of zinc-dependent proteins in newborn Holstein dairy calves. We found that Zn-Met supplementation promoted growth and reduced diarrhea from the second week after birth. It also increased the levels of zinc in the serum and liver, the level of the transporter protein ZIP4 in the jejunal mucosa, as well as the serum alkaline phosphatase and metallothionein concentrations compared to the control group. ZnO supplementation had similar but less marked effects to Zn-Met supplementation. These results suggest that Zn-Met supplementation may be an alternative to antibiotics for the treatment of newborn calf diarrhea.Zinc is considered to be an anti-diarrheal agent, and it may therefore reduce the incidence of diarrhea in young calves. In the present study, we aimed to compare the effect of zinc source on growth performance, the incidence of diarrhea, tissue zinc accumulation, the expression of zinc transporters, and the serum concentrations of zinc-dependent proteins in neonatal Holstein dairy calves. Eighteen male newborn Holstein dairy calves were fed milk and starter diet supplemented with or without 80 mg zinc/d in the form of Zn-Met or ZnO for 14 days, and were then euthanized. Zn-Met supplementation improved average daily gain and feed efficiency, and reduced the incidence of diarrhea, compared with control calves (p < 0.05). It also increased the serum and hepatic zinc concentrations and the mRNA expression of the ZIP4 transporter in the jejunal mucosa of the calves (p < 0.05). In addition, the serum alkaline phosphatase activity and metallothionein concentration were higher in Zn-Met-treated calves than in control calves (p < 0.05). ZnO supplementation had similar effects, but these did not reach significance. Thus, Zn-Met supplementation is an effective means of increasing tissue zinc accumulation and jejunal zinc absorption, and can be used as an anti-diarrheal strategy in neonatal calves.