Abstract: In this study, a vacuum induction furnace (VIF) and a vacuum induction levitation furnace (VILF) were adopted to fabricate 35MnVS steel reinforced with AlTi-MgO nanoparticles, aiming to explore the synergistic regulation mechanism of nanoparticles and smelting processes on inclusions and acicular ferrite (AF) evolution in the steel. The results indicated that the nanoparticle addition significantly refines the inclusions in the steel. Specifically, the average size of inclusions decreases by 30.7% and 70.7% in VIF and VILF steels, respectively. Meanwhile, there was an apparent transition in the composition of inclusions from Al₂O₃-MnS systems to predominantly TiN-MnS composite phases. Due to the effective suppression of nanoparticle agglomeration, the VILF process increases the number density of inclusions per unit area by 210% compared with the conventional VIF process. Thermodynamic and interfacial analyses revealed that TiN-MnS composite inclusions exhibited optimal AF nucleation induction capability (induction rate of 75%) due to their lowest lattice mismatch with ferrite matrix. The synergistic coupling of nanoparticles and the VILF process drives a fundamental microstructural evolution in the steel, resulting in an AF fraction of up to 80.4% and partially replaces the original martensitic phase. This study furnished pivotal theoretical and experimental evidence for the development of high-performance non-quenched and tempered steel via nanoparticle modification and smelting processes optimization.