Abstract Body: Adiponectin, the most abundant circulating adipokine, is important for cardiovascular homeostasis. Our lab previously demonstrated that chronic adiponectin deficiency impairs cardiac and vascular endothelial function. However, the impact of acute adiponectin depletion on endothelial mechanotransduction, including cellular traction forces and flow-mediated dilation (FMD), remains unknown. We hypothesized that acute adiponectin depletion impairs FMD via disruption of sphingosine-1-phosphate (S1P)- and hydrogen peroxide (H₂O₂)-mediated signaling and altered endothelial traction forces, suggesting remodeled mechanosensing in vascular conditions associated with fibrosis and inflammation. Female acute adiponectin knockout (AdipoKO) mice (5-7 months) were generated via tamoxifen inducible Cre-lox recombination along with littermate wild-type (WT) controls of the same genotype without tamoxifen. Gastrocnemius 1A arterioles were isolated and cannulated for assessment of FMD in the absence (control) or presence of N,N-dimethylsphingosine (DMS, a sphingosine kinase inhibitor) or catalase (an H₂O₂ scavenger). Primary aortic endothelial cells from AdipoKO and WT mice were isolated and cultured for traction force microscopy (TFM) analysis on 2 kPa PDMS substrates designed to mimic physiological stiffness (data are Avg. ± SEM). Overall, FMD was reduced in AdipoKO versus WT arterioles (41.6 ± 3.0% vs. 53.1 ± 6.0%; at 10 ΔcmH₂O; n=6-8 mice; p<0.01). In WT arterioles, inhibition of S1P with DMS significantly reduced FMD (18.9 ± 3.1%; Δ-64%; p<0.001), whereas catalase had no significant effect (48.7 ± 3.0%; Δ-18%; p>0.05). In contrast, in AdipoKO arterioles DMS reduced FMD but to a lesser extent than WT arterioles (30.3 ± 3.4%; Δ-27%; p<0.05), and catalase reduced FMD significantly (25.9 ± 1.7%; Δ-38%; p<0.01). Furthermore, traction force microscopy revealed significantly increased endothelial traction forces in AdipoKO versus WT endothelial cells (36.2 ± 3.03 Pa vs. 16.2 ± 1.41 Pa; n=25-48 cells; p<0.001). These findings suggest that adiponectin promotes endothelial biomechanical homeostasis and S1P-dependent FMD under physiological conditions. Acute adiponectin depletion shifts endothelial function toward a mechanically activated phenotype in which FMD becomes more dependent on H₂O₂-mediated signaling. Along with prior evidence from chronic adiponectin deficiency, these results highlight adiponectin as an important regulator of vascular endothelial function.