The comprehensive mechanical behavior of Inconel 625 alloy fabricated by laser powder bed fusion (LPBF) and hot isostatic pressing (HIP) was studied. Tensile experiments were performed at six temperatures ranging from room temperature to 1000 °C to explore the effect of temperature on the mechanical properties, and the microstructure and fracture mechanism was investigated by combining electron backscatter diffraction, transmission electron microscopy, and atom probe tomography. In comparison with the properties of conventionally fabricated Inconel 625 alloys, the mechanical properties at room temperature are essentially the same. However, the plasticity of the Inconel 625 alloys fabricated by LPBF is drastically decreased with increasing temperature, especially when the temperature exceeds 700 °C due to the transformation from transgranular ductile fracture to intergranular fracture. Detailed characterization of cracks and grain boundaries (GBs) reveals significant segregation of non-metallic elements at GBs, which impedes the movement of GBs and decreases their strength, thus enhancing the intergranular embrittlement and resulting in the drastic reduction of ductility. Moreover, the irregular grain morphologies result in numerous GBs directional sharp changes and steps, as well as GBs triple junctions, in which the stress concentration is further enlarged, thus promoting the nucleation of voids and cracks and its associated high temperature embrittlement. The underlying mechanism of ductility deterioration of LPBF-fabricated Inconel 625 alloy at elevated temperatures would provide important insights into its performance at high temperatures.