OBJECTIVE: The aim of this study was to explore the role of pioglitazone (PIO), a peroxisome proliferator-activated receptor-gamma (PPARγ) agonist, in cardiac fibrosis of diabetic mice. MATERIALS AND METHODS: A total of 60 adult male C57/B6 mice were divided into 3 groups using a random number table, namely, control group (Sham group, n=20), diabetic cardiomyopathy group (DCM group, n=20), DCM + PIO group (n=20). Streptozocin (STZ) was injected into mice at a dose of 125 mg/Kg to induce the model of diabetes in vivo. After successful induction, mice in DCM + PIO group were intragastrically given PIO at 10 mg/kg/d once a day for 6 weeks. Meanwhile, those in Sham group and DCM group were given the same volume of normal saline. After 6 weeks, ejection fraction % (EF%), fraction shortening % (FS%) and heart rate of mice in each group were examined via echocardiography. Picrosirius red (PSR) staining assay was conducted to detect collagen deposition in myocardial tissues of mice in each group. The protein expression level of PPARγ in mouse myocardial tissues in each group was measured through Western blotting and immunohistochemical staining assays. Hematoxylin-eosin (H&E) staining assay was carried out to evaluate the myocardial hypertrophy of mice in each group. The protein expression level of transforming growth factor-β (TGF-β) in mouse myocardial tissues in each group was measured through immunohistochemical staining assay. In addition, Western blotting was employed to detect the expression of proteins related to the phosphate and tension homology deleted on chromsome ten (PTEN)/protein kinase B (AKT)/focal adhesion kinase (FAK) signaling pathway in myocardial tissues of mice in each group. RESULTS: The messenger ribonucleic acid (mRNA) and protein expression levels of PPARγ in mouse myocardial tissues were significantly lower in DCM group than those in Sham group (p<0.05). PPARγ agonist PIO could significantly increase the protein expression of PPARγ in myocardial tissues of DCM mice. The results of cardiac Doppler ultrasound revealed that PIO significantly upregulated EF% and FS% in DCM mice (p<0.05). Besides, PIO remarkably reduced collagen deposition and TGF-β protein expression in myocardial tissues in DCM mice (p<0.05). H&E staining results showed that PIO notably attenuated myocardial hypertrophy in DCM mice (p<0.05). Furthermore, it was discovered that PIO markedly elevated PTEN protein in myocardial tissues of DCM mice and inhibited the phosphorylation of AKT and FAK proteins (p<0.05). CONCLUSIONS: The protective effect of PIO against cardiac fibrosis in diabetic mice may be related to its regulation on the PTEN/AKT/FAK signaling pathway. Our findings suggest that PIO is expected to become a targeted drug for the treatment of DCM in clinical practice.