We have previously reported that coronary resistance microvessels (CRMs) from 16‐week‐old type 2 diabetic (db/db) mice were less stiff compared to normal heterozygous Db/db controls, a finding that was driven by less stiff coronary vascular smooth muscle cells (VSMCs). Because the surrounding extracellular matrix (ECM) substrates in vivo may impact cellular properties such as elastic modulus (a measure of stiffness), the aim of this study was to test the hypothesis that modulating substrate stiffness correspondingly modulates the elastic modulus of normal and diabetic coronary VSMCs in vitro. In this study, we plated low‐passage coronary VSMCs isolated from Db/db and db/db mice onto 60 mm polyacrylamide dishes (Matrigen) that were of varying stiffnesses – 4 kPa, 25 kPa, and 50 kPa (n=4 per group). Cells were serum starved for 24 hours, and then cell stiffness was measured by atomic force microscopy (AFM). We observed a stepwise increase in cellular elastic modulus with each increase in polyacrylamide substrate stiffness in both the normal and diabetic coronary VSMCs (Db/db 4 kPa = 3.39±0.48 kPa, Db/db 25 kPa = 5.46±0.78 kPa, Db/db 50 kPa = 7.13±0.29 kPa; db/db 4 kPa = 2.49±0.23, db/db 25 kPa = 5.00±0.52 kPa, db/db 50 kPa = 5.58±0.16 kPa, p<0.0001). Similar to what we have previously reported on cells grown on plastic, diabetic coronary VSMCs were consistently less stiff compared to normal at each substrate stiffness. We did not observe statistically significant differences in cellular filamentous (F) actin, globular (G) actin, or the F/G actin ratio on the varying polyacrylamide substrate stiffness for neither Db/db nor db/db coronary VSMCs. However, there was a preliminary trend for lower F actin (Db/db: 1141.00 ± 472.20 AU vs. db/db: 422.50 ± 240.30 AU at 4 kPa, p=0.24, n=2 per group) and F/G actin ratio (Db/db: 4.01 ± 0.92 AU vs. db/db: 2.55 ± 0.35 AU at 4 kPa, p=0.21, n=2 per group) in the db/db VSMCs, corroborating our past data demonstrating that db/db coronary VSMCs are less stiff compared to normal Db/db coronary VSMCs. Overall, these data show that substrate stiffness correspondingly modulates normal and diabetic coronary VSMC stiffness.