Red blood cells (RBCs) are the most abundant human blood cells. RBC aggregation and deformation strongly determine blood viscosity which impacts hemorheology and microcirculation. In turn, RBC properties depend on different endogenous and exogenous factors. One such factor is nitric oxide (NO), which is mainly produced by endothelial cells (EC) from L-arginine amino acid in the circulatory system. Since the mechanisms of the RBC-endothelium interplay are not clear up to date and considering its possible clinical importance, the aims of this study are to investigate in vitro: (1) The effect of L-arginine induced NO on RBC aggregation and adhesion to endothelium; (2) the NO effect on RBC aggregation and deformation induced by L-arginine and sodium nitroprusside without the presence of endothelium in the samples. The RBC aggregation and adhesion to a monolayer of EC were studied using optical tweezers (OT). The RBC deformability and aggregation without endothelium in the samples were studied using the flow chamber method and Myrenne aggregometer. We confirmed that NO increases deformability and decreases aggregation of RBCs. We showed that the soluble guanylate cyclase pathway appears to be the only NO signaling pathway involved. In the samples with the endothelium, the “bell-shaped” dependence of RBC aggregation force on L-arginine concentration was observed, which improves our knowledge about the process of NO production by endothelium. Additionally, data related to L-arginine accumulation by endothelium were obtained: Necessity of the presence of extracellular L-arginine stated by other authors was put under question. In our study, NO decreased the RBC-endothelium adhesion, however, the tendency appeared to be weak and was not confirmed in another set of experiments. To our knowledge, this is the first attempt to measure the forces of RBC adhesion to endothelium monolayer with OT.