Background: Ischemia/reperfusion (I/R) is a pivotal mechanism of organ injury during clinical stetting for example for cardiopulmonary bypasses. The generation of reactive oxygen species (ROS) during I/R induces oxidative stress that promotes endothelial dysfunction, DNA dissociation and local inflammation. In turn, those processes induce cytokine release, resulting in damage to cellular structures and cell death. One of the major psychoactive compounds of Cannabis is delta-9-tetrahydrocannabinol (ΔΔ99-THC), which is known as an anti-inflammatory mediator. Our research aimed to test if ΔΔ99-THC may be protective in the treatment of cardiovascular system dysfunction arising from I/R heart injury. Methods: Two experimental models were used: isolated rat hearts perfused with the Langendorff method and human cardiac myocytes (HCM) culture. Rat hearts and HCM underwent ex vivo/chemical in vitro I/R protocol with/without ΔΔ99-THC treatment. The following parameters were measured: cell metabolic activity, morphology changes, cell damage as lactate dehydrogenase (LDH) activity, ceramide kinase (CERK) activity, ROS level, total antioxidant capacity (TAC) and heart hemodynamic parameters. Results: ΔΔ99-THC protected the heart, as evidenced by the improved recovery of cardiac function (p << 0.05, N = 3–6). Cells subjected to I/R showed lower cytoplasmic LDH activity, and 10 μ�M ΔΔ99-THC treatment reduced cell injury and increased LDH content (p = 0.019, N = 6–9). Morphology changes of HCM-spherical shape, vacuolisation of cytoplasm and swollen mitochondria—were inhibited due to ΔΔ99-THC treatment. I/R condition affected cell viability, but 10 μ�M ΔΔ99-THC decreased the number of dead cells (p = 0.005, N = 6–9). The total level of CERK was lower in the I/R group, reflecting oxidative/nitrosative stress changes. The administration of ΔΔ99-THC effectively increased the production of CERK to the level of aerobic control (p = 0.028, N = 6–9). ROS level was significantly decreased in I/R cells (p = 0.007, N = 6–8), confirming oxidative stress, while administration of 10 μ�M ΔΔ99-THC enhanced TAC in cardiomyocytes subjected to I/R (p = 0.010, N = 6–8). Conclusions: ΔΔ99-THC promotes the viability of cardiomyocytes, improves their metabolic activity, decreases cell damage and restores heart mechanical function, serving as a cardioprotective. We proposed the use of ΔΔ99-THC as a cardioprotective drug to be, administered before onset of I/R protocol.