The adipose tissue extracellular matrix (ECM) regulates adipocyte cellular metabolism and is altered in obesity and type 2 diabetes, but mechanisms underlying ECM-adipocyte metabolic crosstalk are poorly defined. Advanced glycation end-product (AGE) formation is increased in diabetes. AGE alter tissue function via direct effects on ECM and by binding scavenger receptors on multiple cell types and signaling through Rho GTPases. Our goal was to determine the role and underlying mechanisms of AGE in regulating human ECM-adipocyte metabolic crosstalk. Visceral adipocytes from diabetic and non-diabetic humans with obesity were studied in 2D and 3D-ECM culture systems. AGE is increased in adipose tissue from diabetic compared to non-diabetic subjects. Glycated collagen 1 and AGE-modified ECM regulate adipocyte glucose uptake and expression of AGE scavenger receptors and Rho signaling mediators, including the DIAPH1 gene, which encodes the human Diaphanous 1 protein (hDia1). Notably, inhibition of hDia1, but not scavenger receptors RAGE or CD36, attenuated AGE-ECM inhibition of adipocyte glucose uptake. These data demonstrate that AGE-modification of ECM contributes to adipocyte insulin resistance in human diabetes, and implicate hDia1 as a potential mediator of AGE-ECM-adipocyte metabolic crosstalk.