Background and Purpose Intravascular injection of mesenchymal stem cells (MSCs) has been found to cause considerable vascular obstructions which may lead to serious outcomes, particularly after intra-arterial injection. However, the underlying mechanisms have been poorly understood. Methods In this study, we fractionated MSCs that had been cultured in monolayer for six passages into small (average diameter = 17.9 μm) and large (average diameter 30.4 μm) populations according to their sizes, and examined their vascular obstructions after intra-internal carotid artery injection in rats and mice in comparison with MSCs derived from 3D spheroids which were uniformly smaller in size (average diameter 12.6 μm). Results We found that 3D MSCs did not cause detectable infarct in the brain as evidenced by MRI scan and TTC stain, 2D MSCs in small size caused a microinfarct in one of five animals, which was co-localized to the area of entrapped MSCs (labeled with DiI), while 2D MSCs in large size caused much larger infarcts in all five animals, and substantial amounts of DiI-positive MSCs were found in the infarct. Meanwhile, corresponding neurological defects were observed in the animals with stroke. In consistence, injection of 2D MSCs (average diameter 26.5) caused a marked loss of cortical neurons and their axons in Thy1-GFP transgenic mice and the activation of microglia in CX3CR1-GFP transgenic mice in the area with MSC entrapment. Conclusions Our results suggest that the size of MSCs is a significant cause of MSC caused vascular obstructions and stroke.