Early intestinal development is essential for chicken embryos to fulfill their maximal growth potential. Mannan oligosaccharide (MOS) is known to improve gut morphology, function, and innate immunity; therefore, we hypothesized that its administration in the prehatch period to the sterile intestine of embryos would affect intestinal development and functionality without mediation of gut microflora. The MOS was administered by in ovo feeding procedure to embryos 3 d before hatch. the effects of MOS administration on intestinal morphology, activity of the brush-border enzymes amino peptidase (AP) and sucrase isomaltase (SI) and mRNA abundance of AP, SI, sodium-dependent glucose cotransporter 1 (SGLT1), peptide transporter 1 (PepT1), secreted mucin (MUC2), and toll-like receptors (TLR2 and TLR4) were examined and compared with saline-injected and noninjected controls. Results show that on embryonic d 20 the only parameter affected was MUC2 mRNA abundance, which exhibited a 3-fold increase in the MOS group versus controls. On day of hatch more parameters were affected: a 20 to 32% increase in villus area was found in the MOS group compared with controls; crypt depth and number of goblet cells per villus were higher by 20 and 50%, respectively, compared with the saline group; and AP and SI activities were higher by 44 and 36%, respectively, compared with the noninjected control. In addition, an increase in fold change mRNA abundance of AP, SI, and TLR4 was observed in the MOS group compared with controls. However, on d 3 posthatch, a decrease in MOS effects was noted, indicating a temporally limited effect after administration of 1 dose. In ovo administration of MOS prehatch resulted in a hatching chick with more mature enterocytes and enhanced epithelial barrier and digestive and absorptive capacity at day of hatch. Results imply that the mechanism underlying the observed changes is not mediated through gut microflora but rather involves a direct effect of MOS on intestinal cells.