Estrogen has been closely associated with breast cancer. Several studies reported that Ca2+ signal and Ca2+ channels act in estrogen-modulated non-genomic pathway of breast cancer, however little was revealed on the function of L-type Ca2+ channels. The L-type Ca2+ channel subunit α 1D, named Cav1.3 was found in breast cancer cells. We aimed to investigate the expression and activity of Cav1.3 in human breast cancer, and reveal the effect of estrogen in regulating the expression of Cav1.3. The qRT-PCR and western blotting were employed to show that Cav1.3 was highly expressed in breast cancer tissues. E2 exposure rapidly upregulated the expression of Cav1.3 in dosage- and time-dependent manner, and promoted Ca2+ influx. The silencing of G protein-coupled estrogen receptor 30 (GPER1/GPR30) using siRNA transfection inhibited the upregulation of Cav1.3 and Ca2+ influx induced by E2. Moreover, the inhibition of Cav1.3 by siRNA transfection suppressed E2-induced second peak of Ca2+ signal, the expression of p-ERK1/2, and the cell proliferation. Ultrasound-targeted microbubble destruction (UTMD) of Cav1.3 siRNA was used in MCF-7 cells in vitro and in the tumor xenografts mice in vivo. The application of UTMD significantly suppressed the tumor growth and promoted the survival rate. In conclusion, E2 upregulated the expression of Cav1.3 for Ca2+ influx to promote the expression of p-ERK1/2 for cell proliferation. The study confirmed that the mechanism of E2 inducing the expression of Cav1.3 through a non-genomic pathway, and highlighted that UTMD of Cav1.3 siRNA is a powerful promising technology for breast cancer gene therapy.