Viral infections are described as modifying host gene expression; however, there is limited insight regarding rotavirus (RV) infections. This study aimed to assess the changes in intestinal gene expression after RV infection in a preclinical model, and the effect of 2-fucosyllactose (2′-FL) on this process. From days 2 to 8 of life, rats were supplemented with the dietary oligosaccharide 2′-FL or vehicle. In addition, an RV was inoculated on day 5 to nonsupplemented animals (RV group) and to 2′-FL-fed animals (RV+2′-FL group). Incidence and severity of diarrhea were established. A portion from the middle part of the small intestine was excised for gene expression analysis by microarray kit and qPCR. In nonsupplemented animals, RV-induced diarrhea upregulated host antiviral genes (e.g., Oas1a, Irf7, Ifi44, Isg15) and downregulated several genes involved in absorptive processes and intestinal maturation (e.g., Onecut2, and Ccl19). The 2′-FL-supplemented and infected animals had less diarrhea; however, their gene expression was affected in a similar way as the control-infected animals, with the exception of some immunity/maturation markers that were differentially expressed (e.g., Ccl12 and Afp). Overall, assessing the expression of these key genes may be useful in the evaluation of the efficacy of nutritional interventions or treatments for RV infection.