Hypercontractility of arterial myocytes and enhanced vascular tone during diabetes are, in part, attributed to the effects of increased glucose (hyperglycemia) on L-type Ca_V1.2 channels. In murine arterial myocytes, kinase-dependent mechanisms mediate the increase in Ca_V1.2 activity in response to increased extracellular glucose. We identified a subpopulation of the Ca_V1.2 channel pore-forming subunit (α1_C) within nanometer proximity of protein kinase A (PKA) at the sarcolemma of murine and human arterial myocytes. This arrangement depended upon scaffolding of PKA by an A-kinase anchoring protein 150 (AKAP150) in mice. Glucose-mediated increases in Ca_V1.2 channel activity were associated with PKA activity, leading to α1_C phosphorylation at Ser¹⁹²⁸. Compared to arteries from low-fat diet (LFD)–fed mice and nondiabetic patients, arteries from high-fat diet (HFD)–fed mice and from diabetic patients had increased Ser¹⁹²⁸ phosphorylation and Ca_V1.2 activity. Arterial myocytes and arteries from mice lacking AKAP150 or expressing mutant AKAP150 unable to bind PKA did not exhibit increased Ser¹⁹²⁸ phosphorylation and Ca_V1.2 current density in response to increased glucose or to HFD. Consistent with a functional role for Ser¹⁹²⁸ phosphorylation, arterial myocytes and arteries from knockin mice expressing a Ca_V1.2 with Ser¹⁹²⁸ mutated to alanine (S1928A) lacked glucose-mediated increases in Ca_V1.2 activity and vasoconstriction. Furthermore, the HFD-induced increases in Ca_V1.2 current density and myogenic tone were prevented in S1928A knockin mice. These findings reveal an essential role for α1_C phosphorylation at Ser¹⁹²⁸ in stimulating Ca_V1.2 channel activity and vasoconstriction by AKAP-targeted PKA upon exposure to increased glucose and in diabetes.