Experiments were carried out to determine the effect of cooling rate on the nucleation, growth and microstructure evolution during the solidification process in Ce containing S31254 high Mo austenite stainless steel (HMASS). In situ observations and in situ quenching tests indicate that the Ce effects in this steel are to refine the equiaxed grain and change in the secondary phase from σ to δ, but are mainly suitable for the medium and low cooling rate. The high cooling rate is highly beneficial for austenite nucleation and significantly influence the primary austenite grain growth behavior. The growth velocity for austenite grain is increased with the increase in cooling rate. The morphology of the primary austenite would change from a crescent shape to rod shape with the cooling rate increased from 50°C/min to 100°C/min. Rod-shaped grains with high nucleation densities would make primary grains easy to be combined together, which would reduce the ability of the refined grain. Furthermore, a high cooling rate could slightly improve the solute redistribution, while it significantly deteriorated the micro-segregation of Mo in a solidified structure. The main reason is that high cooling rate could worsen the kinetic condition of ferrite precipitation and postpone the precipitation reaction at the end of the solidification process.