To evaluate the clinic safety of human Midkine as an articular protective agent, recombinant mouse Midkine (rmMK) was prepared in prokaryotic system for the pre-clinic long-term studies in mice. The open reading frame of mouse Midkine (mMK) was sub-cloned onto expression vector pET30a (+) and transformed into Escherichia coli BL21 (DE3) strain line. The rmMK protein, with a Met fused at N terminus of native mMK for expression initiating, proved to be expressed in inclusion bodies and turned out to be soluble post-denaturation and renaturation. The soluble rmMK was purified successfully with ion exchange and affinity chromatography and characterised good enough to meet the requirements for animal use. Eventually, 13.2-mg rmMK with high quality and bioactivity was obtained from 1 L LB culture, and the total recovery was 11.4 %. The present work laid a good foundation for pilot-or large-scale production of rmMK in prokaryotic system.