This work investigated the mechanisms of microstructure and stress-rupture properties evolution of a directionally solidified (DS) Ni-based superalloy, which was thermally exposed at 900 °C up to 1000 h. Microstructure evolution, including the evolution of γ′ phase size, morphology, area fraction and the decomposition of primary MC carbide, was studied during long-term thermal exposure (LTTE). The variation tendency of γ′/γ lattice misfit during LTTE was analyzed. And also, the stress-rupture properties and rupture fractures were tested and observed. The results showed that γ′ phase size increased with the prolongation of thermal exposure time, and the kinetic of γ′ phase coarsening conformed well to Lifshitz-Slyozov-Wagner theory. γ' phase morphology had a tendency to be spheroidized, which was related to the lattice misfit decreasing with the thermal exposure. Meanwhile, the decrease of lattice misfit was the result of redistributed alloy elements during LTTE. The diffusion of carbon from carbide into γ matrix, and the diffusion of Ni, Cr and Co in the opposite direction led to the primary MC decomposition during LTTE. The evolution of stress-rupture life during LTTE was attributed to comprehensive effects of lattice misfit decrease, the evolution of γ' phase size, area fraction and morphology and discrete M23C6 precipitates along grain boundaries.