Acute diarrheal disease (ADD) caused by rotavirus (RV) contributes significantly to morbidity and mortality in children under five years of age. Currently, there are no specific drugs for the treatment of RV infections. Previously, we reported the anti-rotaviral activity of the protein metabolites derived from Bifidobacterium adolescentis. In this study, our aim was to assess the impact of B. adolescentis-secreted proteins (BaSP), with anti-rotaviral activity on the human intestinal C2BBe1 cell line. We initiated the production of BaSP and subsequently confirmed its anti-rotaviral activity by counting the infectious foci using immunocytochemistry. We then exposed the C2BBe1 cells to various concentrations of BaSP (≤250 µg/mL) for 72 h. Cell viability was assessed using the MTT assay, cell monolayer integrity was monitored through transepithelial electrical resistance (TEER), and cytoskeleton architecture and tight junctions (TJs) were examined using confocal microscopy with F-actin and occludin staining. Finally, we utilized a commercial kit to detect markers of apoptosis and necrosis after 24 h of treatment. The results demonstrated that BaSP does not have adverse effects on C2BBe1 cells. These findings confirm that BaSP inhibits rotavirus infectivity and has the potential to strengthen intestinal defense against viral and bacterial infections via the paracellular route.