Protein Kinase Novel 1 (PKN1) is a serine/threonine kinase involved in several cellular functions. PKN1 has been shown to be a stress-responsive kinase acting downstream of small GTP-binding proteins of the Rho/Rac family and 3-phosphoinositide-dependent kinase 1 (PDK1). The role of PKN1 in cardiac cells is still however unclear. In this study, neonatal rat ventricular cardiomyocytes (NRVM) exposed to simulated ischaemia and reperfusion (SI/R) were used as an in vitro model to investigate the role of PKN1 in these cells. shRNA-harbouring adenoviral expression vectors were designed to either silence PKN1 expression (Ad_shRNAPKN1) or to encode a scramble DNA sequence (Ad_shRNAScramble). 60 min of simulated ischaemia followed by 2 h of reperfusion led to a greater increase (2-fold) in cell damage (measured by the bio-reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) and a greater decrease (5-fold) in cell viability (measured by creatine kinase release) in cells infected with Ad_shRNAPKN1 as compared to uninfected cells (p < 0.001) and cells infected with Ad_shRNAScramble (p < 0.05) under the same conditions of SI/R. In preliminary experiments, Human Embryonic Kidney 293 cells overexpressing FLAG^®-tagged PKN1 were subjected to SI/R to gain mechanistic information. Immunoprecipitation of FLAG(R)-tagged PKN1 demonstrated a complex with endogenous PDK1 but not RhoA or Rac1 during SI/R. The findings suggest a cardioprotective role for PKN1 in NRVMs during SI/R. The mechanism by which PKN1 is exerting this effect is yet unknown. The functional significance of the PKN1-PDK1 interaction is being studied as well as its potential downstream substrates.