Pre-endothelialization of a tissue-engineered vascular graft before implantation aims to prevent thrombosis and immunoreactions. This work demonstrates a standardized cultivation process to build a confluent monolayer with human aortal endothelial cells on xenogenous scaffolds. Pre-tested dynamic cultivation conditions in flow slides with pulsatile flow (1 Hz) representing arterial wall conditions were transferred to a newly designed multi-featured rotational bioreactor system. The medium was thickened with 1% methyl cellulose simulating a non-Newtonian fluid comparable to blood. Computational fluid dynamics was used to estimate the optimal volume flow and medium distribution inside the bioreactor chamber for defined wall-near shear stress levels. Flow measurements were performed during cultivation for constant monitoring of the process. Three decellularized porcine arteries were seeded and cultivated in the bioreactor over six days. 1% MC turned out to be the optimal percentage to achieve shear stress values ranging up to 10 dyn/cm2. Vascular endothelial cells formed a continuous monolayer with significant cell alignment in the direction of flow. The presented cultivation protocol in the bioreactor system thus displays a promising template for graft endothelialization and cultivation. Therefore, establishing a key step for future tissue-engineered vascular graft development with a view towards clinical application.