We tested if treatment with sulodexide, a glycocalyx‐mimetic, restores glycocalyx dimensions and whole‐body glucose tolerance induced by a high fat diet (HFD) in mice.C57Bl/6 mice were fed normal chow (n=5) or a HFD for 6 weeks (n=16), and half of these HFD‐fed mice were treated with sulodexide in their drinking water (15mg/100mL) for the final two weeks of the feeding period. At the day of experiment, the microcirculation of the hindlimb muscle was measured with a Sidestream Dark‐field camera under anesthesia, and dynamic variations in RBC column width in all visible microvessels were determined. Further, glucose tolerance was measured by i.p. injection of 1g/kg glucose and determining the area under the curve of blood glucose (AUCgluc).While median perfused RBC column diameter (± 6 μm) did not change, HFD mice were associated with increased outward movement of RBCs into glycocalyx (2.5 ± 0.3 μm vs 1.6 ± 0.3 μm in control, P<0.05), and this effect was impaired after sulodexide treatment (2.1 ± 0.3 μm; NS vs control). The AUCgluc was also increased in HFD‐fed mice versus control (1005.2 ± 59.8 mmol/L*90min. vs 773.7.1 ± 55.0 mmol/L*90min, P<0.05) and this effect was prevented after sulodexide treatment (780.4 ± 31.8 mmol/L*90min., NS vs control).In conclusion, two week sulodexide treatment partially restores glycocalyx dimensions and appears to reverse the HFD‐induced decline in whole‐body glucose tolerance in mice.Supported by DFN 2006.00.027, and CTMM work package 01C‐ 104‐04‐PREDICCT