This study aimed to evaluate the immunomodulatory effect of the probiotic Limosilactobacillus reuteri L26 BiocenolTM (L26) and its purified exopolysaccharide (EPS) with respect to antiviral innate immune response. In our experiment, we used porcine epithelial IPEC-J2 cells as a model of the intestinal barrier in a homologous infection by porcine Rotavirus A strain OSU6 (RVA). The production of selected molecules of non-specific humoral immunity was evaluated at the mRNA level. The EPS alone significantly increased the level of interferon λ3 (IFN-λ3) mRNA in the non-infected IPEC-J2 cells (P < 0.001). We also tested whether the treatment of IPEC-J2 cells by L26 or EPS influences the replication of RVA by virus titration and real-time PCR. We found that a pre-treatment in combination with subsequent continuous stimulation has no influence on the RVA replication. However, both treatments significantly decreased the RVA-induced production of IFN-λ3 (P < 0.05) and the "SOS" cytokine interleukin 6 (IL-6; P < 0.01), already at the transcription level. In addition, the EPS treatment resulted in significantly increased IL-10 mRNA in the RVA-infected cells. In summary, we assume an immunoregulatory potential of L. reuteri L26 BiocenolTM and its EPS in the local intestinal antiviral immune response.