<p>A truncated VP4* fragment of Bovine Rotavirus was expressed in Escherichia coli, and optimal conditions for recombinant protein isolation and purification were established. Comprehensive bioinformatics-driven in silico and in vivo evaluation of the VP4* protein was carried out to assess its structural and immunological properties. In silico analysis of physicochemical properties revealed that VP4*fragments are antigenic, non-toxic, and non-allergenic. Structural validation showed that 99.3% of residues were within the favored region of the Ramachandran plot, confirming the reliability of the 3D model structure of the protein. The recombinant protein with bovine Toll-like Receptors (TLRs) was validated by molecular docking via ClusPro, showing strong binding affinities between the complexes. Molecular dynamics simulations (Desmond software) and computational immunology (C-ImmSim server) consistently indicated the constructs' structural integrity, stable receptor interaction, and capacity to elicit robust cellular and humoral immune responses. The in vivo immunogenicity results of VP4*antigen in Wistar rats were validated by the results obtained from computational immunological analysis. A comparative immune response study of the VP4* antigen with the rotavirus vaccine (ROTAVAC) or as a booster dose after vaccination was observed in Wistar rats. A high IgG level was shown in the group where the antigen was given as a booster dose after vaccination with no side effects in the rats, indicating that the protein is non-toxic, non-allergenic, potentially immunogenic, and represents a promising candidate for further evaluation in rotavirus prevention.</p> Graphical Abstract <p></p>