Effect of isothermal holding treatment in the solidification process on the microstructure of Ti-6Al-4V alloy was studied by temperature controlled induced melting apparatus. The result shows that with isothermal holding treatment above the β transus temperature during solidification, the colony structure consisting of parallel lamellae was obtained. While the isothermal holding treatment was set at 960 °C, a unique bi-modal microstructure consisting of coarse primary α and fine secondary lamellar α was obtained. The primary lamellar α tended to break into several pieces, globularize and present equiaxed morphology. The formation mechanism of the equiaxed α can be explained with the atom immigration, high density dislocations, combined action with the interface tension of formed α phase during the isothermal holding treatment. After the isothermal holding, the retained β matrix transformed into fine lamellar α, thus, bi-modal microstructure was acquired. Compared with the lamellar structure, the grain boundary α presented discontinuously and cannot be distinguished from the primary α lamellae easily. The size of colonies α was greatly decreased. The microstructure tended to be much more homogeneous in the whole section of the samples.