In light of recent developments in nanotechnologies, interest is growing to better comprehend the interaction of nanoparticles with body tissues, in particular within the cardiovascular system. Attention has recently focused on the link between environmental pollution and cardiovascular diseases. Nanoparticles <50 nm in size are known to pass the alveolar–pulmonary barrier, enter into bloodstream and induce inflammation, but the direct pathogenic mechanisms still need to be evaluated. We thus focused our attention on titanium dioxide (TiO2) nanoparticles, the most diffuse nanomaterial in polluted environments and one generally considered inert for the human body. We conducted functional studies on isolated adult rat cardiomyocytes exposed acutely in vitro to TiO2 and on healthy rats administered a single dose of 2 mg/Kg TiO2 NPs via the trachea. Transmission electron microscopy was used to verify the actual presence of TiO2 nanoparticles within cardiac tissue, toxicological assays were used to assess lipid peroxidation and DNA tissue damage, and an in silico method was used to model the effect on action potential. Ventricular myocytes exposed in vitro to TiO2 had significantly reduced action potential duration, impairment of sarcomere shortening and decreased stability of resting membrane potential. In vivo, a single intra-tracheal administration of saline solution containing TiO2 nanoparticles increased cardiac conduction velocity and tissue excitability, resulting in an enhanced propensity for inducible arrhythmias. Computational modeling of ventricular action potential indicated that a membrane leakage could account for the nanoparticle-induced effects measured on real cardiomyocytes. Acute exposure to TiO2 nanoparticles acutely alters cardiac excitability and increases the likelihood of arrhythmic events.