Microstructure variation of Ti6Al4V alloy after hydrogenation was investigated by optical microscopy (OM), X-ray diffraction (XRD) and transmission electron microscopy (TEM), and the formation mechanism of hydride was discussed. The influence of hydrogen content on hardness of Ti6Al4V alloy was studied by hardness testing. The result shows that hydrogen could reduce (��+��)/�� transformation temperature obviously. Hydride �� (fcc structure) as well as orthorhombic martensite �� precipitated in the specimens after hydrogenation, and hydrides precipitated not only from �� phase but also from �� phase. It is considered in this paper that the formation of �� in �� phase is a phase transformation based on the diffusion of hydrogen, and the formation of �� in �� phase is the result of eutectoid reaction �� H�����+��. The result of hardness testing shows that the hardness of Ti6Al4V alloy increases after hydrogenation, which is caused by formation of �� hydrides, hydrogen solution strengthening, interaction between hydrogen and dislocation and martensitic transformation jointly with increasing of hydrogen.