The voltage gated L-type calcium channels (LTCCs) are the main pathway for Ca²⁺ influx in vascular smooth muscle cells (VSMCs). They are critical for excitation-contraction coupling and have recently been shown also to be important in excitation-transcription coupling. VSMCs exist in different phenotypes, which can be characterized as contractile and non-contractile or proliferative. Switch between these phenotypes is associated with altered expression of LTCCs. In this study we investigated whether reduced expression of LTCC in vivo can induce the phenotypic switch. We downregulated LTCCs with siRNA against the a1c subunit in rat small mesenteric arteries in vivo. Artery structure and function were tested in vitro 3 days later. The arteries neighboring the LTCC downregulated arteries were transfected with non-related siRNA and used as controls. In downregulated arteries LTCC expression (mRNA) was 24±5% (n=6) of control. Active media wall stress in response to K⁺-induced depolarization was reduced (to ~16%, n=10) as was the increase of intracellular Ca²⁺ (to ~24%, n=7). The force development to caffeine was also attenuated while Ca²⁺ rose similar to the control. a-toxin permeabilized, downregulated arteries also had reduced force development suggesting that LTCC downregulated VSMCs lose the ability to contract despite an increase in Ca²⁺. The expression (mRNA) of marker genes of the contractile phenotype was significantly reduced and marker genes for proliferation upregulated in LTCC downregulated arteries. LTCC downregulation was associated with an increase in media thickness, caused by an increase number of VSMCs per segment length. We demonstrated here that LTCCs are crucial in determining VSMC phenotype as well as the structure of resistance arteries.