To determine the effects of chloride channel 3 (ClC-3) knockdown and overexpression on lysophosphatidic acid (LPA)- and volume-regulated anion channel Cl− currents ( ICl,LPA and ICl,VRAC, respectively), cell differentiation, and cell volume regulation, a short hairpin RNA (shRNA) expression system based on a mouse U6 promoter was used to knock down ClC-3 in human corneal keratocytes and human fetal lung fibroblasts. ClC-3 overexpression was achieved by electroporating full-length ClC-3, within a pcDNA3.1 vector, into these two cell lines. RT-PCR and Western blot analysis were used to detect ClC-3 mRNA and protein levels. Whole cell perforated patch-clamp recording was used to measure ICl,LPA and ICl,VRAC currents, and fluorescence-activated cell sorting analysis was used to measure cell volume regulation. ClC-3 knockdown significantly decreased ICl,LPA and ICl,VRAC activity in the presence of transforming growth factor-β1 (TGF-β1) compared with controls, whereas ClC-3 overexpression resulted in increased ICl,LPA activity in the absence of TGF-β1. ClC-3 knockdown also resulted in a reduction of α-smooth muscle actin (α-SMA) protein levels in the presence of TGF-β1, whereas ClC-3 overexpression increased α-SMA protein expression in the absence of TGF-β1. In addition, keratocytes transfected with ClC-3 shRNA had a significantly blunted regulatory volume decrease response following hyposmotic stimulation compared with controls. These data confirm that ClC-3 is important in VRAC function and cell volume regulation, is associated with the ICl,LPA current activity, and participates in the fibroblast-to-myofibroblast transition.