In recent years, (0001) twist grain boundaries (BTGBs) located in primary α grain clusters were identified as fatigue crack nucleation sites in different Ti alloys. In the present study, crack initiation was investigated in a bimodal Ti-5Al-4V alloy subjected to low-cycle fatigue and dwell-fatigue loadings at room temperature. The low fraction of primary α grains was not associated with a lack of sensitivity to BTGB cracking. Transmission electron microscopy and electron back-scattered diffraction were used to characterize BTGBs in the initial microstructure. The fatigue mechanisms were then analyzed with a focus on dislocation activity. αp grains adjacent to cracked BTGBs contained a high dislocation density. It was primarily composed of planar slip bands of <a> dislocations. In addition, <c+a> dislocations were noticed in the vicinity of cracked BTGBs. They supposedly pertain to crack tip plasticity during growth, and no evidence of a role of an incoming slip event in crack nucleation was obtained. Also, basal slip bands extending across adjacent grains were found to emerge from BTGBs. This feature provides an easier path for crack extension when growth along the grain boundary becomes difficult owing to a deviation from the basal plane. Atom probe tomography analyses evidenced V and Fe segregation at a grain boundary with a significant deviation from the BTGB configuration. This suggests a possible contribution of local solute segregation to the high cracking resistance of general αp / αp grain boundaries. This work provides new insights into the mechanisms involved in cracking of BTGB in Ti alloys subjected to cyclic loadings.