Proton pump inhibitors (PPIs) are widely prescribed for acid-related disorders and are generally considered safe for short-term use. However, increasing experimental and clinical evidence suggests potential cardiovascular effects associated with chronic exposure, possibly related to endothelial dysfunction and impaired nitric oxide bioavailability. Therefore, we decided to investigate whether the cardiovascular effects of omeprazole are dependent on the timing of administration in a model of cardiac ischemia-reperfusion (CIR) in normotensive Wistar rats (NWR) and spontaneously hypertensive rats (SHR). Twelve- to sixteen-week-old male NWR and SHR were allocated into four groups: (1) SHAM: NWR and SHR were submitted to surgery with no ischemia; (2) (SS + CIR): NWR and SHR were treated with a 0.9% saline solution and submitted to CIR; and (3) (OME + ISQ): NWR and SHR were treated with 10 mg/kg i.v. omeprazole (OME) before cardiac ischemia and submitted to CIR or (4) after cardiac ischemia but before cardiac reperfusion (ISQ + OME). Electrocardiograms were monitored to assess ventricular arrhythmias (VA), atrioventricular block (AVB), and lethality (LET). Serum creatine kinase-MB (CK-MB) levels were quantified, and histopathological analyses were performed to evaluate the degree of myocardial injury in the different study groups. Administration of OME prior to cardiac ischemia increased the incidence of VA, AVB, LET, and serum CK-MB levels in both NWR and SHR. In contrast, administration before cardiac reperfusion did not exacerbate cardiac injury and was associated with the attenuation of electrophysiological instability. Histopathological findings corroborated the biochemical and functional outcomes. OME, when administered prior to cardiac ischemia, worsens both cardiac arrhythmias and myocardial injury; however, administration immediately prior to cardiac reperfusion does not increase cardiac arrhythmias and decreases myocardial injury in both NWR and SHR.