Aims: Type 4 collagen encoded by COL4A1 gene is an integral component of blood-brain barrier (BBB). It has been reported that mutations in COL4A1 can cause various abnormalities in human cerebral microvasculature. We sought to determine the pathological consequences of COL4A1 deficiency in brain microvessels of adult mice. Methods: To knock out COL4A1 gene specifically in brain endothelial cells (BECs) of adult mice, we combined an adeno-associated virus to target the brain endothelium (AAV-BR1) with CRISPR/Cas9 system. The gRNA targeting exon 1 of COL4A1 was selected, packaged with AAV-BR1, and injected into Cas9 transgenic mice. The mice were sacrificed and COL4A1 levels in BECs measured 3 weeks after injection. At 3 months, the mice were monitored by MRI and sacrificed to examine the histopathology in the brain. Results and Conclusions: AAV-BR1-COL4A1 gRNA labeled with mCherry was primarily detected in BECs in vitro and in vivo. We observed 97% out-of-frame deletion of COL4A1 gene and marked downregulation of COL4A1 level. Unexpectedly, T2*-weighted gradient echo imaging revealed that multiple low-intensity signals with a diameter less than 0.5 mm were diffusely observed in different areas of brain, strikingly resembling the cerebral microbleeds described in human patients. Also, two-photon/electron microscopy imaging and histopathological examination implied that microhemorrhage and hemosiderin-laden microglia/macrophages were detected in disrupted BBB. This study indicates that a deficiency of type 4 collagen can cause structural disintegration of brain microvessels leading to microhemorrhages. Furthermore, our results suggest that human microbleeds may be linked to dysfunction of type 4 collagen in the BBB.