HS, a complex sugar, is synthesised onto heparan sulfate proteoglycan (HSPG) core proteins and is essential for islet beta cell survival. T2D-prone db/db mice are characterised by obesity, insulin resistance, and ultimately beta cell failure. Our previous studies showed a significant loss of HS and HSPG core proteins in islets of T2D-prone db/db mice by 6 weeks of age, compared to wildtype (wt) controls. This study investigated whether HS replacement can prevent T2D beta cell death in vitro.The blood glucose (bg) of female wt and db/db donor mice (5-8 weeks of age) was measured using a glucometer. Wildtype and db/db islets were isolated from donor pancreases and dispersed into single cells using Accutase (250μl/500 islets). The beta cells were cultured with or without heparin (50μg/ml) for 2 days and beta cell viability was analysed by flow cytometry using the fluorescent dyes calcein (Cal) and propidium iodide (PI). For glucose tolerance tests, wt and db/db mice were fasted for 6 hrs, glucose (2g/kg body weight) was injected intraperitoneally and blood glucose levels were measured at 0, 15, 30, 60, 90 and 120 minutes.After culture with heparin, wt beta cells showed a 3.5-fold increase in viable Cal⁺PI^- beta cells, compared to untreated controls (P=0.0001). Heparin-treated db/db beta cells from donors with bg<10mmol/L and bg=10-15mmol/L showed a significant 2.5-fold (P=0.0001) and 2.2-fold (P=0.01) increase in viability, respectively. No significant change was observed in the viability of heparin-treated beta cells from donors with bg>15mmol/L. Impaired glucose tolerance was observed in db/db mice, but not wt mice.Our findings demonstrate that in vitro HS replacement can protect the viability of beta cells from normoglycaemic and mildly hyperglycaemic (bg<15mmol/L) db/db mice. We propose that HS mimetics may represent a new class of therapeutic for preserving beta cell survival and function in T2D.