Background: Novel sweetening ingredients with potential for high dietary exposure require comprehensive genotoxicity assessment as a cornerstone of safety evaluation. Objective: To evaluate the mutagenic potential of crystalline 5-keto-D-fructose (5-KDF) using a bacterial reverse mutation assay conducted according to OECD Test Guideline 471 scientific principles. Methods: Plate-incorporation assays were performed using five tester strains (Salmonella typhimurium TA98, TA100, TA1535, TA1537, and Escherichia coli WP2 uvrA) in the absence (−S9) and presence (+S9) of an exogenous metabolic activation system. 5-KDF was tested at concentrations of 50, 150, 500, 1,500, and 5,000 μg/plate in triplicate, alongside vehicle controls (deionized water) and strain-appropriate positive controls. Acceptance criteria included concurrent negative control values within laboratory historical control ranges and robust positive control responses confirming assay sensitivity. Results: No precipitation or cytotoxicity was observed at any tested concentration up to the guideline limit dose of 5,000 μg/plate. Revertant colony counts for all 5-KDF treatment groups were comparable to vehicle controls across all five strains under both −S9 and +S9 conditions, with no concentration-related increases or reproducible elevations. Positive controls elicited strong, expected mutagenic responses in all strains, confirming test system competence. Conclusion: Under the conditions tested, 5-keto-D-fructose did not induce reverse mutations in the bacterial reverse mutation assay up to the OECD TG 471 limit dose of 5,000 μg/plate.