In order to investigate the relationship between microstructure and MnS inclusion in non-quenched and tempered steel, and cooling rate during horizontal directional solidification, 49MnVS steel was used to conduct the experiments with a self-designed device. The mathematical effect of cooling rate on dendritic arm spacing and mean diameter of MnS particles (dMnS) were determined by using linear regression method. The results show that the length of dendrite from solid–liquid interface to end-solidification decreased with increasing the withdrawal velocity (ϑ). dMnS has a similar value in the area of the steady directional solidification; the value of dMnS was 4.1, 3.6, 3.3, 2.8 and 2.3 μm at withdrawal velocity of 50, 75, 100, 150 and 200 μm/s, respectively. dMnS increased with reducing ϑ or RC (interface cooling rate). MnS precipitated in the gaps between dendrites and was influenced by secondary dendritic arm spacing. Besides, a new concept of the ‘Precipitation Unit Space’ (PUS) was proposed and the relationships between dMnS, VPUS (volume of PUS) and RC were obtained.