Administration of insulin is recommended to patients with severe sepsis and hyperglycemia. Previously we demonstrated that insulin may have direct anti-inflammatory properties and counteracted fluid losses from the circulation by normalizing the interstitial fluid pressure (P_IF). P_IF is one of the Starling forces determining fluid flux over the capillary wall and a lowered PIF is one of the driving forces in early edema formation in inflammatory reactions. Here we demonstrate that insulin restores a lipopolysaccharide (LPS)-lowered P_IF via a mechanism involving integrin V3. In C57black mice (n=6), LPS lowered PIF from -0.2 ± 0.2 mmHg to -1.6 ± 0.3 mmHg (p<0.05) and after insulin averaged -0.8 ± 0.2 mmHg (p=0.098 compared to after LPS). Corresponding values in wild type BALB/c mice (n=5) were -0.8 ± 0.1 mmHg, -2.1 ± 0.3 mmHg (p<0.05) and -0.8 ± 0.3 mmHg (p<0.05 compared to LPS) after insulin administration. In BALB/c integrin 3 deficient (3-/-) mice (n=6) LPS lowered P_IF from -0.1 ± 0.2 mmHg to -1.5 ± 0.3 mmHg (p<0.05). Insulin did, however, not restore P_IF in these mice (averaged -1.7 ± 0.3 mmHg after insulin administration). Cell-mediated collagen gel contraction can serve as an in vitro model for in vivo measurements of P_IF. Insulin induced V3 integrin-dependent collagen gel contraction mediated by C2C12 cells. Our findings suggest a beneficiary effect of insulin for patients with sepsis with regard to the fluid balance, and this effect may in part be due to a normalization of P_IF by a mechanism involving the integrin V3.