Stevioside, a natural glycoside compound, has many beneficial biological activities, but its protective effect on myocardial fibrosis has not been reported yet. This study aimed to investigate the effect of stevioside. The isoproterenol-induced model mice were orally given stevioside 75-300 mg kg-1 for 40 days. The results showed that after the administration of stevioside, the myocardial hydroxyproline, collagen accumulation, and protein expressions of collagen I/III, α-smooth muscle actin, transforming growth factor-β1 (TGF-β1), nuclear factor kappa B p65 (NF-κB p65), Smad2/3, and P-Smad2/3 were decreased, while the glutathione peroxidase and superoxide dismutase levels in serum and myocardial tissues and protein expressions of myocardial peroxisome proliferator-activated receptor γ (PPARγ) and Smad7 were increased. After the preincubation of isoproterenol-stimulated cardiac fibroblasts with the PPARγ antagonist GW9662, stevioside-reduced protein expressions were decreased, but stevioside-induced Smad7 was not affected. These findings demonstrated that stevioside could exert a protective effect on mouse myocardial fibrosis, and its mechanisms were associated with the increments of antioxidant ability, PPARγ activation, and Smad7 expression, which caused a synergistic inhibition of the NF-κB/TGF-β1/Smad signaling pathway.