To determine the effects of ClC-3 knockdown and overexpression on LPA- and volume-regulated 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 utilized to knockdown 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 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 FACS analysis was used to measure cell volume regulation. ClC-3 knockdown significantly decreased ICl_LPA and ICl_VRAC activity in the presence of TGF-1 compared to controls, whereas ClC-3 overexpression resulted in increased ICl_LPA activity in the absence of TGF-₁. ClC-3 knockdown also resulted in a reduction of -SMA protein levels in the presence of TGF-₁, whereas ClC-3 overexpression increased -SMA protein expression in the absence of TGF-1. In addition, keratocytes transfected with ClC-3 shRNA had a significantly blunted RVD response following hyposmotic stimulation compared to 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.