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Precipitation of MnS inclusions in steel affects the mechanical properties of the material significantly. The evolution of MnS inclusions along the continuous casting slab thickness and its influencing factors has not been clearly established and comprehensively studied. In this paper, solidification macrostructure, sulfur segregation and MnS inclusions in the continuous casting slab of medium carbon structural steel 45# were studied by various methods, including the metallographic observations, elemental analysis, scanning electron microscope (SEM) with Energy Dispersive Spectrometer (EDS) observation, automatic particle analysis, and thermodynamic calculations. The 2D/3D morphologies of MnS inclusions suggest that the sulfides turn from globular to rodlike, and further to dendritic shape along the slab thickness progressively. Furthermore, it was found that MnS inclusions are remarkably aggregated in the columnar crystals and the equiaxed crystals mixed zone, where the sulfides have the largest average diameter of 6.35 μm and the second maximum area fraction of 0.025% along the slab thickness. In order to reveal the mechanism of this phenomenon, the precipitation temperature of MnS inclusion in the 45# steel was clarified by thermodynamic calculation and experimental observation, and the quantitative relationships among the distribution of sulfur content, secondary dendrite arm spacing (SDAS), and precipitation area fraction of MnS inclusions were discussed. Moreover, the inclusion size was numerically predicted to compare with the measured value. The results indicate that the large SDAS, high sulfur content and low cooling rate accounting for the large-size aggregated MnS inclusions in the mixed zone. Unfortunately, the dendritic MnS inclusions, even if the average diameter exceeds 52 μm, can act as the nucleation sites for ferrites, and the distribution of the sulfides promotes uneven microstructure in the steel.