Blood flow velocity profile in microvessels is essential for in vivo studies of substance exchange between blood and tissue. This paper was aimed to investigate the temporal and spatial variations in the velocity profile of red blood cell (RBC) flow in arterioles with both bifurcation and confluence in the rat mesentery, using a particle image velocimetry (PIV). The microcirculation in rat mesentery was observed under a microscopic system with a high-speed digital camera. The images of RBCs flow in microvessels were recorded simultaneously with the arterial blood pressure. Based on the high-speed video images, instantaneous velocity vectors of RBCs in arterioles with bifurcation and confluence were evaluated using a high spatial-resolution PIV algorithm. Then, the time-averaged and ensemble-averaged velocity profiles over the cross-section were calculated from the proximal through the bifurcation to the distal to the confluence. The velocity profile of RBCs showed two peaks at bifurcation and confluence, respectively. The double peaks were most marked at the apex of bifurcation, but not so much marked at the confluence. The variation of centerline velocity showed that the length of vessel under the influence of bifurcation or confluence, was approximately 1.5 times the diameter at the proximal to the apex of bifurcation, but its length was reduced significantly at the distal of confluence.