Vinculin and its splice isoform metavinculin play key roles in regulating cellular morphology， motility， and force transduction. Vinculin is distinct from metavinculin in its ability to bundle filamentous actin (F-actin). To elucidate the molecular basis for these differences， we employed computational and experimental approaches. Results from these analyses suggest that the C terminus of both vinculin and metavinculin form stable interactions with the F-actin surface. However， the metavinculin tail (MVt) domain contains a 68 amino acid insert， with helix 1 (H1) sequestered into a globular subdomain， which protrudes from the F-actin surface and prevents actin bundling by sterically occluding actin filaments. Consistent with our model， deletion and selective point mutations within the MVt H1 disrupt this protruding structure， and facilitate actin bundling similar to vinculin tail (Vt) domain.