The aims of this study were to investigate the effect of Saccharomyces cerevisiae var. boulardii CNCM I-1079 (SCB) or Lactobacillus acidophilus BT1386 (LA) on (1) innate immune response, (2) markers of acute-phase reaction, and (3) immune gene expression of rumen and ileum tissues of Holstein calves. Forty eight calves (?5 d old) were randomly allocated to four treatments as follows: (1) control (CTRL) fed milk replacer followed by starter feed, (2) CTRL supplemented with SCB in milk and feed, (3) CTRL supplemented with LA in milk and feed, and (4) CTRL supplemented with antibiotics (ATB; chlortetracycline and neomycin in milk, and chlortetracycline in feed). Tumor necrosis factor α (TNF-α) decreased (P < 0.05) on day 66 (post-weaning) for the ATB-treated calves. There were no treatment effects on production of interferon γ (IFN-γ) and interleukin 6 (IL-6) proteins and on expression of TLR4, TLR6, TLR9, TLR10, CLDN3, MUC1, and MUC20 genes. Calves fed SCB or LA had a greater (P < 0.05) oxidative burst at weaning (day 53) compared with CTRL. Oxidative burst was also greater (P < 0.05) after weaning (day 59 and day 87) for SCB-fed calves. Calves fed SCB and ATB had higher (P < 0.05) phagocytosis activity during weaning (day 47) compared with CTRL. The concentration of serum amyloid A2 (SAA2) increased (P < 0.05) in SCB- and LA-fed calves (day 53), whereas the concentration of C-reactive protein (CRP) increased (P < 0.05) in SCB-fed calves during weaning as compared with CTRL. Our results suggest that SCB could improve innate immune response (oxidative burst and phagocytosis) and markers of acute-phase reaction (CRP and SAA2), especially during critical periods like weaning.