Due to their high glucose permeability, insulin-secreting pancreatic beta-cells likely undergo strong intracellular protein glycation at high glucose concentrations. They may, however, be partly protected from the glucotoxic alterations of their survival and function by fructosamine-3-kinase (FN3K), a ubiquitous enzyme that initiates deglycation of intracellular proteins. To test that hypothesis, we cultured pancreatic islets from Fn3k-knockout (Fn3k(-/-)) mice and their wild-type (WT) littermates for 1-3 wk in the presence of 10 or 30 mmol/l glucose (G10 or G30, respectively) and measured protein glycation, apoptosis, preproinsulin gene expression, and Ca(2+) and insulin secretory responses to acute glucose stimulation. The more potent glycating agent d-ribose (25 mmol/l) was used as positive control for protein glycation. In WT islets, a 1-wk culture in G30 significantly increased the amount of soluble intracellular protein-bound fructose-epsilon-lysines and the glucose sensitivity of beta-cells for changes in Ca(2+) and insulin secretion, whereas it decreased the islet insulin content. After 3 wk, culture in G30 also strongly decreased beta-cell glucose responsiveness and preproinsulin mRNA levels, whereas it increased islet cell apoptosis. Although protein-bound fructose-epsilon-lysines were more abundant in Fn3k(-/-) vs. WT islets, islet cell survival and function and their glucotoxic alterations were almost identical in both types of islets, except for a lower level of apoptosis in Fn3k(-/-) islets cultured for 3 wk in G30. In comparison, d-ribose (1 wk) similarly decreased preproinsulin expression and beta-cell glucose responsiveness in both types of islets, whereas it increased apoptosis to a larger extent in Fn3k(-/-) vs. WT islets. We conclude that, despite its ability to reduce the glycation of intracellular islet proteins, FN3K is neither required for the maintenance of beta-cell survival and function under control conditions nor involved in protection against beta-cell glucotoxicity. The latter, therefore, occurs independently from the associated increase in the level of intracellular fructose-epsilon-lysines.