Abstract: Mo-Re alloys are promising candidates for advanced nuclear reactor components and space power systems owing to their high-temperature strength, irradiation resistance, and compatibility with nuclear fuels. However, the intrinsic low-temperature brittleness of body-centered cubic (BCC) Mo and the unclear microscopic mechanism of Re-induced softening have long limited the predictive design and wider application of these alloys. To investigate the microscopic mechanism of Re-induced softening in Mo-based alloys, molecular dynamics simulations of nanoindentation were performed on pure Mo and Mo-14Re (14%Re) alloy. A spherical rigid indenter with a radius of 4 nm was pressed into the substrate to a maximum depth of 4 nm. Thirty independent simulations were carried out for each case to ensure statistical reliability. The interatomic interactions were described by a recently developed Mo-Re potential. The load-displacement curves were recorded, and the hardness was calculated with correction for pile-up effects. Dislocation nucleation and evolution were analyzed using dislocation extraction algorithms, and the activation volume for dislocation nucleation was determined from pop-in events. The effect of temperature was examined by repeating the simulations at 300 K, 700 K, and 1 100 K. The results show that both the indentation load and the hardness of Mo-14Re are consistently lower than those of pure Mo, confirming a clear Re softening effect. At 300 K, the hardness of pure Mo is (3.29±0.25) GPa, whereas that of Mo-14Re alloy is (3.11±0.28) GPa, representing a reduction of approximately 5.5%. The activation volume for dislocation nucleation increases from 20.5 ų in pure Mo to 22.1 ų in Mo-14Re alloy, indicating that the dislocation nucleation in Mo-14Re alloy involves complex cooperative migration of several atoms. Meanwhile, the dislocation density in Mo-14Re alloy remains lower than that in pure Mo throughout the indentation process. In both materials, dislocation evolution is dominated by 〈111〉-type dislocations (accounting for about 90% of the total dislocation density), followed by 〈100〉-type dislocations (about 8%). During indentation, independent dislocation loops are observed to form through the expansion and connection of U-shaped dislocation arms along the 〈111〉 directions, a mechanism analogous to the “Lasso” process reported in other BCC metals. When the temperature is raised from 300 K to 700 K and then to 1 100 K, the hardness of Mo-14Re alloy decreases progressively to (2.48±0.86) GPa and (1.46±0.07) GPa, respectively, accompanied by a reduction in dislocation density. A positive correlation between hardness and dislocation density is observed: higher dislocation density leads to stronger dislocation interactions and entanglement, which in turn increases resistance to plastic deformation. In conclusion, the addition of 14%Re softens the Mo alloy by increasing the activation volume for dislocation nucleation and reducing the dislocation density. Temperature elevation further weakens the alloy by promoting dislocation annihilation. These atomic-scale insights provide a new perspective for understanding the Re effect in BCC refractory alloys and offer guidance for the design of high-performance Mo-based materials.