This study focused on exploring the ability of self‐assembled DNA frameworks to cross the blood‐brain barrier (BBB). We designed and assembled a series of DNA origami structures with equal quantity of nucleic acid materials but different morphologies and rigidities, such as barrel, soccer ball, icosahedron, and compared their transport efficiency in an in vitro BBB model. It was observed that the relatively large and soft structures could better penetrate the BBB through a lysosome irrelative transcytosis process, while the smallest and most rigid structure was blocked severally accompanied with an obvious lysosome digestion once internalized by the endothelial cells. Three framework DNA origami structures with equal molecular weight but different shape, size and rigidity were compared in parallel to investigate their penetration behaviors cross an artificial blood‐brain barrier and potential mechanisms.