The specific gravity of titanium alloy is 4.5 which is lighter than steel, however, its specific strength, heat and corrosion resistance are superior to steel. Therefore, it is used in spaceships, medicines, and so on. Ti-6Al-4V alloy is a kind of α+β phase titanium alloy, and mechanical properties are changed by alloy elements, shapes and distributions of microstructures. In this paper, the effect of microstructure on mechanical behavior for Ti-6Al-4V alloy was studied. Two different kinds of specimens are prepared under different heat treatments (rolled plate, 1050℃) in order to produce different microstructures. Various kinds of mechanical tests such as hardness, tensile, fatigue and fretting fatigue tests are performed for evaluation of mechanical properties with the changes of microstructures. Through these tests, the following conclusions are observed: 1) Microstructures are observed as equiaxed and widmanstatten microstructures respectively. 2) Impact absorbed energy is superior for the equiaxed microstructure, and the hardness and tensile strength are superior for the widmanstatten microstructure. 3) The fatigue endurance of widmanstatten microstructure shows higher value than that of the equiaxed microstructure. 4) The fatigue endurance in fretting condition was reduced about 50% from that of the non-fretting condition.