High glucose (HG) in peritoneal dialysates has been demonstrated to induce extracellular matrix (ECM) synthesis by peritoneal mesothelial cells (PMCs) and to contribute to peritoneal fibrosis during continuous ambulatory peritoneal dialysis (CAPD). In the present study, we investigated the effects of pioglitazone, a peroxisome proliferator-activated receptor γ (PPARγ) agonist, on HG-induced ECM accumulation and the underlying mechanism in rat PMCs (RPMCs). In cultured RPMCs, HG treatment increased the expression of fibronectin (FN), collagen I and plasminogen activation inhibitor-1 (PAI-1) at the mRNA and protein levels, while it downregulated the expression of PPARγ in a time- and concentration-dependent manner. Pretreatment with pioglitazone not only decreased the expression of PAI-1 and matrix proteins (FN and collagen I), but prevented the downregulation of PPARγ in RPMCs under HG conditions. HG treatment activated the nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) pathways. In addition, the NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), and the AP-1 inhibitor, SP600125, decreased the protein levels of FN, collagen I and PAI-1, suggesting a role for the NF-κB and AP-1 pathways in the regulation of ECM accumulation induced by HG in RPMCs. Notably, we demonstrated that pretreatment with pioglitazone significantly inhibited HG-induced NF-κB and AP-1 activation. Collectively, these results suggest that pioglitazone inhibits HG-induced ECM accumulation in RPMCs by increasing PPARγ expression, and by inhibiting the NF-κB and AP-1 pathways.