Three dimensional atom probe (3DAP) combined with SEM and TEM was applied to characterize the alloying elements partitioning segregation in cementite in experimental steel tempering at 450℃ for 4h after solution treatment at 1200℃ for 0.5h. The results indicated that, in the as-quenched specimen nearly all the alloying elements such as manganese, silicon and vanadium were dissolved in matrix with homogeneous distribution, and a slight segregation of carbon for the sake of autotempering. A significant segregated zone of carbon found in the 450℃ tempering specimen was detected as a cementite, in which was there an enrichment of manganese, a slight segregation of molybdenum and vanadium and negligible aluminum content, silicon was prone to partitioning at cementite/matrix interface from cementite. A monolayer in thickness enriched with carbon and vanadium was also detected, which may be a V4C3 based G.P. zone acted as an initiator of precipitation of alloyed carbides during an intensity or extensive tempering.