Objective The present study investigated the effect of microRNA-328 (miR-328) during the aerobic exercise-induced improvement of voltage-gated L-type Ca2+ channel ( LTCC) expression in thoracic aorta smooth muscle cells from spontaneously hypertensive rats ( SHRs). Methods Twelve-week-old male normotensive Wistar Kyoto rats (WKYs) and SHRs randomly were separated into a control group (SHR-C and WKY-C) and an exercise group (SHR-EX and WKY-EX). Rats in exercise were subjected to moderate-intensity treadmill training for 12 weeks. The cardiovascular responses of rats were then detected by femoral arterial and venous cannulation. The thoracic aorta was stained with hematoxylin and eosin ( HE) for histological assays. The mRNA expression levels of LTCC α_1C and β₁ subunits and the expression of miR-328 were detected by qPCR. The protein expression levels of LTCC α_1C and β₁ subunits were detected by Western Blot. For in vitro analysis, primary vascular smooth muscle cells (VSMCs) were isolated and cultured from the thoracic aorta of WKYs. Both miR-328 mimic and miR-328 inhibitor were transfected into cultured arterial myocytes to produce miR-328 overexpression or silencing. The mRNA and protein expression levels of LTCC α 1C and β₁ subunits were detected. Results Aerobic exercise significantly reduced body weight, blood pressure, and heart rate in SHRs. Aerobic exercise significantly reduced the wall thickness of thoracic aorta in SHRs. Exercise significantly reduced the pressor response to norepinephrine and the depressor response to nifedipine ( LTCC blocker) in SHRs. The mRNA and protein expression levels of LTCC α_1C and β₁ subunits were significantly upregulated in the SHR-C group, compared with the WKY- C group, while the mRNA and protein expression levels of LTCC α_1C and β₁ subunits were downregulated in the SHR-EX group, compared with the SHR-C group. miR-328 expression was significantly lower in the SHR-C group than that in the WKY-C group; this expression significantly increased after aerobic exercise. In the transfection experiment, compared with the negative control ( NC) group, the mRNA and protein expression levels of α_1C and β₁ subunits were significantly downregulated in the miR-328 mimic transfected group; these expression was significantly upregulated in the miR-328 inhibitor transfected group. Conclusions Regular aerobic exercise may effectively reduce blood pressure in SHR and enable miR-328 to inhibit LTCC α_1C and β₁ subunit expression. This mechanism might contribute to exercise-mediated improvement of LTCC channel remodeling in hypertensive thoracic aorta.