Objective To investigate the effects of 2,2,6,6-tetramethyl-4-piperidinol (tempol) on NF-κB signaling pathway of myocardial hypertrophy in rats. Methods The rat model of myocardial hypertrophy was established by intraperitoneal injection of isoprenaline (ISO) (5 mg/ kg, twice per day, 2 weeks). A total of 42 male SD rats were randomly divided into 3 groups, including the control group, myocardial hypertrophy model group (ISO + sterile saline) and tempol treatment group (ISO + tempol) [tempol 100 mg/ (kg·d), 8 weeks]. Eight weeks after the corresponding drug intervention, the heart weight index (HWI) and left ventricular weight index (LVWI) were determined. Morphology and fibrosis of the myocardium were observed by HE staining, and the myocardial fibrosis of the rats was observed by Masson staining. The mRNA levels of TNF-α and IL-6 in the rat myocardial tissues were detected by qRT-PCR, and the expression levels of IκBα, p-p65 and p65 were detected by Western blot. Results Compared with the control group, the HWI and LVMI, mRNA levels of TNF-α and IL-6, and expression of p-p65/ p65 in the model group were significantly increased (P < 0.05), while the expression level of IκBα, an NF-κB inhibitory protein was significantly decreased (P <0.05). The pathological examination of the myocardial tissues showed thickening and disordered arrangement of myocardial fibers, and increased cross-sectional area of the myocardial fibers. The pathology by Masson staining showed aggravated myocardial fibrosis and increased collagen fibers in the myocardial interstitium. Compared with the model group, the HWI and LVMI, the mRNA levels of TNF-α and IL-6, and the expression of p-p65/ p65 of the tempol group were significantly decreased (P < 0.05), while the expression level of IκBα was significantly increased (P < 0.05). HE staining showed that the degrees of myocardial disarrangement and cardiomyocyte hypertrophy were decreased. Meanwhile, Masson staining showed that the extent of myocardial fibrosis was reduced, and the interstitial collagen fibers were decreased. Conclusions Tempol can improve the isoprenaline-induced myocardial hypertrophy, which may be closely related with the inhibition of the activity of NF-κB signaling pathway.