Flagellin is a major component of the flagellar filament. Flagellin also functions as a specific ligand that stimulates innate immunity through direct interaction with Toll-like receptor 5 (TLR5) in the host. Because flagellin activates the immune response, it has been of interest to develop as a vaccine adjuvant in subunit vaccines or antigen fusion vaccines. Despite the widespread application of flagellin fusion in preventing infectious diseases, flagellin-antigen fusion designs have never been biophysically and structurally characterized. Moreover, flagellin from Salmonella species has been used extensively despite containing hypervariable regions not required for TLR5 that can cause an unexpected immune response. In this study, flagellin from Bacillus cereus (BcFlg) was identified as the smallest flagellin molecule containing only the conserved TLR5-activating D0 and D1 domains. The crystal structure of BcFlg was determined to provide a scheme for fusion designs. Through homology-based modeling and comparative structural analyses, diverse fusion strategies were proposed. Moreover, cellular and biophysical analysis of an array of fusion constructs indicated that insertion fusion at BcFlg residues 178-180 does not interfere with the protein stability or TLR5-stimulating capacity of flagellin, suggesting its usefulness in the development and optimization of flagellin fusion vaccines.