Aims: The aim of this study was to determine the antiviral activity of four probiotic metabolites (Lactobacillus and Bifidobacetrium species) against rotavirus in vitro infection monitored by the NSP4 protein production and Ca 2+ release. Methods and Results: The antiviral effect of the metabolites was performed due a comparison between a blocking model and an intracelullar model on MA104 cells, with the response of NSP4 production and Ca 2+ liberation measured by flow cytometry. Significant results were obtained with the metabolites of Lactobacillus casei, and Bifidobacterium adolescentis in the reduction of the protein production (P = 0Á04 and P = 0Á014) and Ca 2+ liberation (P = 0Á094 and P = 0Á020) in the intracellular model, which suggests a successful antiviral activity against RV infection. Conclusions: This study demonstrates that probiotic metabolites were able to interfere with the final amount of intracellular NSP4 protein and a successful Ca 2+ regulation, which suggests a new approach to the mechanism exerted by probiotics against the rotavirus infection. Significance and Impact of the Study: A novel anti-rotaviral effect exerted by probiotic metabolites monitored by the NSP4 protein during the RV in vitro infection and the effect on the Ca 2+ release is reported; suggesting a reduction on the impact of the infection by decreasing the damage of the cells preventing the electrolyte loss.