This study investigates the low-cycle fatigue behavior and microstructural evolution of a novel 30Cr2Ni3MoWV hot-work die steel at 700 °C under different strain amplitudes. High-temperature tensile tests demonstrated a tensile strength of 460 MPa and an elongation of 32%, confirming the material retains good ductility. Fracture analysis revealed ductile failure, supported by a 95% reduction in area. Low-cycle fatigue tests indicated notable cyclic softening at high strain amplitudes, with fatigue life declining rapidly as strain amplitude rose from 0.2% to 0.6%. A stress-softening coefficient model was established to describe this accelerated softening. Microstructural examination identified carbides (MC, M₇C₃, M₂₃C₆), which promoted secondary crack formation at 0.6% strain amplitude, contributing to early failure. TEM analysis further showed dislocation rearrangement, carbide coarsening, and martensite lath widening during cyclic loading. Among these, M₂₃C₆ precipitates were linked to increased softening at higher strains. The Coffin–Manson model parameters were optimized based on the relationship between fatigue life, plastic strain, and elastic strain. The model accurately predicted the steel’s fatigue life, with only a 0.01% deviation from experimental results. This work correlates accelerated softening and reduced fatigue life with three microstructural mechanisms—carbide coarsening, dislocation accumulation, and secondary cracking—offering valuable guidance for enhancing the high-temperature performance of hot-work die steels.