Poloxamer (P)188 is a tri-block copolymer shown to provide a cardioprotective effect when administered at the beginning of reoxygenation in adult mouse cardiomyocyte hypoxia/reoxygenation models. It is thought that it seals cells damaged by hypoxia via membrane stabilization, thus, protecting the cells from additional injury during reoxygenation. To further explore the effects of P188 on maintaining endothelial cell layer integrity, we treated confluent adult mouse coronary artery endothelial cell layers with varying concentrations of P188 upon initiation of a 2-hr reoxygenation period immediately following hypoxic periods of 3, 12, and 48 hrs. Using trans-endothelial electrical resistance (TEER), we found that P188 dose-dependently protected cells from further damage after hypoxia, with some doses returning TEER measurements even back to control/normoxia levels. However, we also observed that similar concentrations of P188 varied in effects as hypoxia times increased. Our results indicate that P188 protects the endothelial layer in a dose-dependent manner as long as it is administered after an optimal ischemia time to effectively preserve membrane integrity in tissue barriers. More research will need to be conducted to confirm our findings and determine the optimal combination of dosage and timing of P188 administration.