Mouse 3T3-L1 preadipocytes are widely used for metabolic study of obesity; however, their cellular physiology is not fully understood. The present study investigates functional ion channels and their role in the regulation of cell proliferation using whole-cell patch voltage-clamp, RT-PCR, Western blot, and cell proliferation assay in undifferentiated 3T3-L1 preadipocytes. We found three types of ionic currents present in 3T3-L1 preadipocytes, including an inwardly-rectifying K+ current (IKir, recorded in 15% of cells) inhibited by Ba2+, a Ca2+-activated intermediate K+ current (IKCa, recorded in 44% of cells) inhibited by clotrimazole (or TRAM-34) as well as a chloride current (ICl) inhibited by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) in 12% of cells, which can be activated in all cells with hypotonic (0.8 T) insult, implicating a volume-sensitive ICl (ICl.vol). RT-PCR and Western blot analysis revealed the expression of KCa3.1 (for IKCa), Kir2.1 (for IKir), and Clcn3 (for ICl.vol). Blockade of IKCa with TRAM-34 or ICl.vol with DIDS inhibited cell proliferation in a concentration-dependent manner. Knockdown of KCa3.1 or Clcn3 with specific siRNAs also suppressed cell proliferation. Flow cytometry analysis showed that blockade or silencing of KCa3.1 or Clcn3 channels with corresponding blockers or siRNAs caused an accumulation of cells at the G0/G1 phase. These results demonstrate that three functional ion channel currents, IKCa, ICl.vol, and IKir, are heterogeneously present in 3T3-L1 preadipocytes. IKCa and ICl.vol participate in the regulation of cell proliferation. J. Cell. Physiol. 227: 1972–1979, 2012. © 2011 Wiley Periodicals, Inc.