The results of an investigation on the low cycle fatigue (LCF) behavior, at room temperature and 400°C for four conventional microstructures (Widmannstatten, basket-weave, equiaxed, and duplex) in a TC6 titanium alloy are presented. The fatigue crack nucleation and propagation in fatigue-tested specimens have been observed by scanning electron microscopy (SEM). The duplex microstructure is associated with the longest LCF life at room temperature and 400°C, while the Widmannstatten microstructure has the shortest. The crack initiation sites and propagation paths were examined and discussed. The cracks primarily initiated along slip bands on the specimen surface for all four microstructures. In addition, many voids appeared along slip bands for the equiaxed microstructure. By linking-up these voids, the formation of microcracks is realized. The propagation of interior cracks in specimens with Widmannstatten structure proceeded by cross-cutting Wα platelets by way of a plastic blunting mechanism, whereas for the equiaxed microstructure interior cracks grew by the linking-up of voids by way of a renucleation mechanism.