When incubated under simulated microgravity (s-mg), endothelial cells (EC) form tubular structures that resemble vascular intimas. This delayed formation of 3D EC structures begins between the 5th and 7th day of culturing EC under conditions of s-mg. With the aim of learning about this initial phase of tubular structure formation, NFkBp65 protein content was similar in all cell populations, but gene and protein expression of phosphokinase A catalytic subunit, phosphokinase Ca, and extracellular signal-regulated kinases 1 and 2 was altered in cells cultured under s-mg. In contrast to controls, the 7-day-old s-mg cultures contained 3D aggregates with proliferating cells, enhanced numbers of necrotic cells, and osteopontin-negative EC as well as supernatants with reduced quantities of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), soluble TNFRSF5, TNFSF5, intercellular adhesion molecule-1, tumor necrosis factor receptor 2, IL-18, complement C3, and von Willebrand factor. VEGF and=or bFGF (10 ng=mL) application influenced the accumulation of proteins in supernatants more profoundly under 1 g than under s-mg. These findings provide evidence that phosphokinase Ca plays a key role in tube formation. Improving the interaction of VEGF and=or bFGF with EC under s-mg could enhance the engineering of vascular intimas.