Invariant natural killer T cells (iNKT) perform different functions in different diseases. The cells were reported to protect myocarditis. However, the detail relationships between iNKT and Coxsackievirus B3 (CVB3)-induced myocarditis remain unclear. In order to investigate the correlation between the severity of CVB3-induced inflammation infiltration and the proportion of iNKT in the spleen and circulating blood, BALB/c mice were grouped into three groups according to the inflammation infiltration area of heart sections. The proportion of iNKT in CD3-positive cells in the spleen correlated negatively with the inflammation area (linear fit; R(2)=0.93) and virus capsid protein VP1 (linear fit; R(2)=0.84) in the myocardial tissue, while the proportion of iNKT in CD3-positive cells in the PBMC positively correlated with the inflammation area (linear fit; R(2)=0.91) and virus capsid protein VP1 (linear fit; R(2)=0.93) in the myocardial tissue. The results imply that iNKT might be used as a parameter for the diagnosis of myocarditis in clinical practice.