Filamin B (FLNB) is a dimeric actin-binding protein that orchestrates the reorganization of the actin cytoskeleton. Congenital mutations of FLNB at the actin-binding domain (ABD) are known to cause abnormalities of skeletal development, such as atelosteogenesis type-I/III and Larsen's syndrome, although the underlying mechanisms are poorly understood. Here, using fluorescence microscopy, we characterized the reorganization of the actin cytoskeleton in cells expressing each of 6 pathological FLNB mutants that have been linked to skeletal abnormalities. The subfractionation assay showed a greater accumulation of the FLNB ABD mutants, W148R and E227K, to the cytoskeleton, compared with the wild type protein. Ectopic expression of FLNB-W148R, and the E227K protein with a lesser extent, induced formation of prominent F-actin accumulations, the consequent rearrangements of focal adhesions, myosin-II and septin filaments, and results in a delayed directional migration of the cells. The W148R protein-induced cytoskeletal re-arrangement was partially attenuated by the inhibition of myosin-II, PAK or ROCK. The expression of a single-head ABD fragment with the mutations partially mimicked the rearrangement induced by the dimer. The F-actin clustering through the interaction with the mutant FLNB ABD may limit the cytoskeletal reorganization preventing normal skeletal development.