This article reports the morphology, size, and distribution evolution of MnS inclusions in non-quenched and tempered steel during heat treatment. The variation of single large-sized MnS inclusions at high temperature was observed in situ using a confocal scanning laser microscope (CSLM). The slender MnS inclusions first changed to pearl-like strings. These small-sized pearls subsequently coalesced and became closer together as the temperature increased. Large-sized MnS inclusions in non-quenched and tempered steel samples with different thermal histories were investigated with respect to the evolution of their morphology, size, and distribution. After 30 min of ovulation at 1573 K, the percentage of MnS inclusions larger than 3 µm decreased from 50.5% to 3.0%. After a 3 h soaking period, Ostwald ripening occurred. Most MnS inclusions moved from the grain boundaries to the interior. The present study demonstrates that heat treatment is an effective method of changing the morphology, size, and distribution of MnS inclusions, especially large-sized ones.