The impact property of high-strength Ti-5Al-7.5V-0.5Si-0.25Fe-0.2O alloy (Ti575) with trimodal microstructure (TM) and lamellar microstructure (LM) were investigated by combining instrumented Charpy U-notch impact test with microstructure characterization. Impact load-displacement curves illustrated that the higher impact toughness of LM (47.8 J/cm2) than TM (38 J/cm2) were attributed to both intrinsic toughening during crack initiation and extrinsic toughening during crack propagation. In crack initiation stage of LM, electron back-scattered diffraction (EBSD) evidenced the nucleation of numerous {10–12} <–1011> tensile twins in coarsened lamellar α (αl) in vicinity of the crack notch. The high intrinsic toughness of LM was due to the dynamic microstructure refinement and facilitation of prismatic/pyramidal slip induced by twins during impact. For TM in contrast, smaller-sized primary α(αp) and αl suppressed the nucleation of twins, exacerbated stress concentration and reduced plastic deformation near the crack notch. In crack propagation stage, the kinking, shearing of αl, crack deflection at αl/β interfaces and secondary crack in αl colony borders contributed to a tortuous crack path and large energy dissipation in LM, whereas crack could bypass αp and cut through thin αl in TM easily, resulting in a flat crack path and poor extrinsic toughness of TM. In summary, this work provided a basic understanding of the impact fracture mechanism of Ti575 with different microstructures.