Background: 5-Keto-D-fructose (5-KDF) is a rare monosaccharide with potential applications as a functional sweetener in food products. Comprehensive in vitro safety assessment is essential for regulatory evaluation under GRAS (Generally Recognized As Safe) frameworks. Objective: To evaluate the cytotoxic potential and mechanistic safety profile of 5-KDF in human-relevant cell models representing primary exposure tissues. Methods: Two human cell lines—Caco-2 (intestinal epithelium) and HepG2 (hepatocytes)—were exposed to 5-KDF across physiologically relevant concentrations (0.1–100 mM) and a supraphysiological concentration (200 mM) for 24 and 72 hours. Cytotoxicity was assessed via ATP-based viability assays (CellTiter-Glo®). Mechanistic endpoints in HepG2 cells included: reactive oxygen species generation (DCFDA), glutathione redox status (GSH/GSSG ratio), mitochondrial membrane potential (JC-1), mitochondrial bioenergetics (Seahorse XF Mito Stress Test), caspase-3/7 activation, and apoptosis/necrosis characterization (Annexin V/PI flow cytometry). Glucose, fructose, D-allulose, and mannitol served as equimolar osmotic comparators; appropriate positive controls validated assay performance. Results: 5-KDF demonstrated no cytotoxicity at concentrations up to 100 mM in both cell models across all exposure durations, with viability maintained at 94.1–101.8% of vehicle controls. At 200 mM, modest viability reductions (88.2–92.1%) were observed but were statistically and quantitatively comparable to equimolar carbohydrate and osmotic controls, consistent with non-specific hyperosmotic stress rather than compound-specific toxicity. Mechanistic evaluations revealed no evidence of oxidative stress induction, mitochondrial dysfunction, or activation of apoptotic/necrotic pathways at physiologically relevant concentrations (≤100 mM). All positive controls elicited expected responses, confirming assay sensitivity and specificity. Conclusions: 5-KDF exhibits no intrinsic cytotoxicity or mechanistic warning signals in human intestinal and hepatic cell models at concentrations spanning predicted dietary exposure levels. The comprehensive negative findings across multiple orthogonal endpoints support the conclusion that 5-KDF does not pose cellular toxicity concerns under anticipated conditions of use as a food ingredient.