Inhibition of translocator protein (18 kDa) (TSPO) can effectively prevent reperfusion-induced arrhythmias and improve postischemic contractile performance. Mitochondrial permeability transition pore (mPTP) opening, mediated mainly through oxidative stress during ischemia/reperfusion (I/R), is a key event in reperfusion injury. 4'-Chlorodiazepam is a widely used TSPO antagonist. However, whether 4'-chlorodiazepam can improve cardiac functional recovery during postischemia reperfusion by affecting oxidative enzymes, reducing reactive oxygen species (ROS) and thereby inhibiting mPTP opening is still unknown. Cardiac function including heart rate, coronary flow rate, left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP), maximal time derivatives of pressure (+/-dP/dt max) and the severity of ventricular arrhythmias were analyzed in isolated rat hearts during I/R. mPTP opening, ROS and oxidative enzyme activities were measured with fluorometric or spectrophotometric techniques. 4'-Chlorodiazepam did not affect heart rate and coronary flow rate, but abolished the increase in LVEDP, accelerated the recovery of LVDP and +/-dP/dt max, and reduced the severity of ventricular arrhythmias. The mPTP opening probability was reduced by 4'-chlorodiazepam, accompanied by a reduction in ROS level. In addition, the activities of mitochondrial electron transport chain complex I and complex III were increased, while those of xanthine oxidase and NADPH oxidase were reduced. Therefore, 4'-chlorodiazepam may improve cardiac functional recovery during reperfusion, potentially by affecting the activities of oxidative enzymes, reducing ROS and thereby inhibiting mPTP opening. The present study presents evidence that 4'-chlorodiazepam could be a novel adjunct to reperfusion.