OBJECTIVE To evaluate the usefulness of intestinal biomarkers in determining the presence of intestinal epithelial damage in neonatal calves with diarrhea caused by 4 etiologic agents. ANIMALS 40 neonatal calves that were healthy (n = 10) or had diarrhea (30). PROCEDURES The study was a cross-sectional study. Results of hematologic analyses and serum concentrations of intestinal fatty acid–binding protein (I-FABP), liver fatty acid–binding protein (L-FABP), trefoil factor 3 (TFF-3), Claudin-3 (CLDN-3), γ-enteric smooth muscle actin (ACTG2), intestinal alkaline phosphatase (IAP), interleukin-8 (IL-8), platelet-activating factor (PAF), and leptin (LP) were compared among calves grouped according to whether they were healthy (control group; G-1) or had diarrhea caused by K99 Escherichia coli (G-2; n = 10), bovine rota- or coronavirus (G-3; 5 each), or Cryptosporidium spp (G-4; 10). RESULTS Across the 3 time points at which blood samples were obtained and evaluated, the groups of calves with diarrhea generally had markedly higher mean serum concentrations of L-FABP, TFF-3, IAP, IL-8, and LP, compared with the control group. In addition, G-2 also consistently had markedly higher mean serum concentrations of I-FAB and ACTG2 and lower mean serum concentrations of CLDN-3, compared with the control group. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that degree of intestinal epithelial damage differed among calves grouped by the etiologic agent of diarrhea and that such damage might have been more severe in calves with diarrhea caused by K99 E coli. Additionally, our results indicated that serum concentrations of I-FABP, L-FABP, TFF-3, IAP, IL-8, ACTG2, LP, and CLDN-3 were useful biomarkers of intestinal epithelial damage in calves of the present study.