Intravenous bolus (IVb) injection of overdose fentanyl triggers an immediate apnea followed by severely depressed ventilation with several associated effects, such as upper airway constriction, respiratory muscle rigidity, hypoxemia/hypercapnia (due to hypoventilation) and hypothermia. This study sought to determine the direct impact of IVb injection of fentanyl on phrenic nerve activity (PN) over time in anesthetized, vagotomized, paralyzed and ventilated rats to avoid the associated effects. The integrative PN (?PN), frequency of ?PN (fR), minute PN (MPN, ?PN × fR), heart rate (HR), arterial blood pressure (ABP), SpO2 and PETCO2 and their responses to hypoxia (10% O2 in nitrogen for 1 min) and then hypercapnia (10% CO2 in 30% O2 and 60% nitrogen for 3 min) were recorded before and after IVb injection of fentanyl (30 µl/kg). Fentanyl induced an extra long-lasting apnea (for 6 min) with an-18 sec latency followed by sustained and stable MPN depression (↓70%), hypertension and tachycardia. Hypoxia (decreasing SpO2 by 30%) augmented MPN associated with slight but significant hypotension and tachycardia. The PN responses were strikingly reduced and the cardiovascular responses exacerbated by fentanyl. Hypercapnia (increasing PETCO2 to 70 torr) also enhanced MPN associated with hypertension and bradycardia, and the MPN responses were blunted with the evoked hypertension turned to hypotension and the bradycardia aggravated after fentanyl. We conclude that fentanyl tremendously reduces the inspiratory motor drive partially via attenuating the chemoreflexes, which is responsible for the ventilatory depression and failure observed in the clinical setting.