Voltage-gated Ca2+ channels (CaV) are ubiquitously expressed in various cell types and play vital roles in regulation of cellular functions including proliferation. However, the molecular identities and function of CaV remained unexplored in preadipocytes. Therefore, whole cell voltage-clamp technique, conventional/quantitative real-time RT-PCR, Western blot, small interfering RNA (siRNA) experiments, and immunohistochemical analysis were applied in mouse primary cultured preadipocytes as well as mouse 3T3-L1 preadipocytes. The effects of CaV blockers on cell proliferation and cell cycle were also investigated. Whole cell recordings of 3T3-L1 preadipocytes showed low-threshold CaV, which could be inhibited by mibefradil, Ni2+ (IC50 of 200 μM), and NNC55-0396. Dominant expression of α1G mRNA was detected among CaV transcripts (α1A–α1I), supported by expression of CaV3.1 protein encoded by α1G gene, with immunohistochemical studies and Western blot analysis. siRNA targeted for α1G markedly inhibited CaV. Dominant expression of α1G mRNA and expression of CaV3.1 protein were also observed in mouse primary cultured preadipocytes. Expression level of α1G mRNA and CaV3.1 protein significantly decreased in differentiated adipocytes. Mibefradil, NNC55-0396, a selective T-type CaV blocker, but not diltiazem, inhibited cell proliferation in response to serum. NNC55-0396 and siRNA targeted for α1G also prevented cell cycle entry/progression. The present study demonstrates that the CaV3.1 T-type Ca2+ channel encoded by α1G subtype is the dominant CaV in mouse preadipocytes and may play a role in regulating preadipocyte proliferation, a key step in adipose tissue development.