We investigated the behavior of red blood cells (RBCs) in a microchannel with stenosis using a confocal micro-PTV system. Individual trajectories of RBCs in a concentrated suspension of up to 20% hematocrit (Hct) were measured successfully. Results indicated that the trajectories of healthy RBCs became asymmetric before and after the stenosis, while the trajectories of tracer particles in pure water were almost symmetric. The asymmetry was greater in 10% Hct than in 20% Hct. We also investigated the effect of deformability of RBCs on the cell-free layer thickness by hardening RBCs using a glutaraldehyde treatment. The results indicated that deformability is the key factor in the asymmetry of cell-free layer thickness. Therefore, the motions of RBCs are influenced strongly by the Hct, the deformability, and the channel geometry. These results give fundamental knowledge for a better understanding of blood flow in microcirculation and biomedical microdevices.