The formation and characteristics of non-metallic inclusions in 304L stainless steel during the vacuum oxygen decarburization (VOD) refining process were investigated using industrial experiments and thermodynamic calculations. The compositional characteristics indicated that two types of inclusions with different sizes (from 1 μm to 30 μm) existed in 304L stainless steel during the VOD refining process, i.e., CaO-SiO2-Al2O3-MgO external inclusions, and CaO-SiO2-Al2O3-MgO-MnO endogenous inclusions. The calculation results obtained using the FactSage 7.1 software confirmed that the inclusions that were larger than 5 μm were mostly CaO-SiO2-Al2O3-MgO; the similarity in composition to the slag indicated that these inclusions originated from the slag entrapment. The CaO-SiO2-Al2O3-MgO-MnO inclusions that were smaller than 5 μm originated mainly from the oxidation reaction with Ca, Al, Mg, Si, and Mn. The changes in the inclusion composition resulting from changes in the Ca, Al, and O contents, and the temperature during the VOD refining process was larger for the smaller inclusions. Generating mechanisms for the CaO-SiO2-Al2O3-MgO-MnO inclusions in the 304L stainless steel were proposed.