Human milk (HM) is rich in oligosaccharides (HMO) that exert prebiotic and anti-infective activities. HM feeding reduces the incidence of rotavirus (RV) infection in infants. Herein, the anti-RV activity of oligosaccharides was tested in an established in vitro system for assessing cellular binding and viral infectivity/replication, and also tested in a newly developed, acute RV infection, in situ piglet model. For the in vitro work, crude HMO isolated from pooled HM, neutral HMO (lacto-N-neotetraose, LNnT; 2′-fucosyllactose) and acidic HMO (aHMO, 3′-sialyllactose, 3′-SL; 6′-sialyllactose, 6′-SL) were tested against the porcine OSU strain and human RV Wa strain. The RV Wa strain was not inhibited by any oligosaccharides. However, the RV OSU strain infectivity was dose-dependently inhibited by sialic acid (SA)-containing HMO. 3′-SL and 6′-SL concordantly inhibited 125I-radiolabelled RV cellular binding and infectivity/replication. For the in situ study, a midline laparotomy was performed on 21-d-old formula-fed piglets and six 10 cm loops of ileum were isolated in situ. Briefly, 2 mg/ml of LNnT, aHMO mixture (40 % 6′-SL/10 % 3′-SL/50 % SA) or media with or without the RV OSU strain (1 × 107 focus-forming units) were injected into the loops and maintained for 6 h. The loops treated with HMO treatments+RV had lower RV replication, as assessed by non-structural protein-4 (NSP4) mRNA expression, than RV-treated loops alone. In conclusion, SA-containing HMO inhibited RV infectivity in vitro; however, both neutral HMO and SA with aHMO decreased NSP4 replication during acute RV infection in situ.