Isothermal heat treatment can not only modify steel microstructure, but also non-metallic inclusions. In this work, heat treatment experiments were conducted between 1373 and 1573 K (1100 and 1300 °C) to study the evolution of inclusion composition, morphology, and size distribution. Results showed that during the heat treatment at 1473 and 1573 K (1200 and 1300 °C), two main kinds of inclusions initially in the steel, CaS and MgO–Al2O3–CaO–CaS, gradually transformed to (Ca, Mn)S and MgO–Al2O3–(Ca, Mn)S inclusions, and some MgO–Al2O3–CaO inclusions also transformed to MgO–Al2O3–(Ca, Mn)S. At the lowest temperature studied, 1373 K (1100 °C), little change was observed. No significant changes in number density and area fraction of the measured inclusions were observed, while the average size of inclusions increased after the heat treatment. The extent of transformation of CaS, MgO–Al2O3–CaO–CaS and MgO–Al2O3–CaO inclusions increased with decreasing inclusion size and higher temperature.