Doxorubicin (DOX) is an effective chemotherapy treatment for various cancers, but its uses due to cardiotoxicity effects have been limited. There are rare studies about the protective effect of HIIT against DOX-induced cardiac toxicity and its mechanisms. The purpose of this study was to investigate the protective effect of the HIIT against the DOX-induced cardiotoxicity by examining the expression levels of miR-499, widely expressed in the cardiomyocytes under the physiological conditions. 24 male Wistar rats were randomly assigned into four groups (n=6/groups): 1) Control; 2) DOX (20 mg/kg body weight); 3) HIIT (7 sets of 4 min intervals at 80%–90% VO2max interspersed with 3 min periods of 65%–75% VO2max); and 4) HIIT + DOX. The mRNA levels were determined using RT-PCR. Statistical differences were analyzed using the one-way analysis of variance followed by Tukey's post hoc test for multiple comparisons (α<0.05). Based on the results, DOX-treatment significantly increased the expression of miR-499 in the left ventricular tissue of rats (P<0.05). Also, the expression of miR-499 increased after exercise, but this difference was not significant. Exercise before the DOX-induction also significantly reduced the expression of miR-499 in the HIIT+ DOX group compared to the DOX group (P<0.05). Therefore, the HIIT could be a proper strategy for protecting the heart against ADR-induced cardiotoxicity via reducing miR-499 expression.