Recent studies show that long-term cocaine use induces diastolic impairment. This functional effect may be linked to cocainetriggered biochemical responses such as oxidative stress. Indeed, previously we have demonstrated that myocardial NADPH and xanthine oxidase (XO) contribute to ROS generation in cocainetreated rats. In the current study, we hypothesized that cocaineinduced ROS production could induce mitochondrial damage that in turn might participate in ventricular diastolic dysfunction. Wistar rats were treated with cocaine alone (2×7.5mg/kg/day, IP) or with a NOX inhibitor (apocynin, 50mg/kg/day, po) or a XO inhibitor (allopurinol, 50mg/kg/day, po). These groups were compared to control rats (saline solution, IP). After 7 days, LV pressure-volume signals were acquired. Oxygen consumption was measured in situ on permeabilized cardiac fibers isolated from the LV. ROS production was measured using electron paramagnetic resonance in both subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria. Our results show that cocaine-induced cardiac dysfunction is characterized by a diastolic impairment. Indeed cocaine induces an increase of Tau, an index of LV relaxation and of end-diastolic pressure volume relation (+80 % and +171 % respectively, p<0.05). Both apocynin and allopurinol were able to improve ventricular relaxation. Further, we found that cocaine increased oxygen consumption in mitochondria specifically through complex I (+34 %, p<0.05) and complex III (+120 %, p<0.05) whereas ATP production was decreased. Moreover, ROS level increased only in IFM from cocaine rats (+74 %, p<0.05). In contrast, apocynin or allopurinol treatments prevented the rise in ROS levels and prevented the mitochondrial respiratory chain alterations. In conclusion, this work shows that cocaine-induced LV diastolic impairment is associated with a specific mitochondrial dysfunction. An increase in ROS production via NADPH and XO induces a mitochondrial dysfunction which in turn contributes to the development of oxidative stress and ventricular diastolic dysfunction.