Dickkopf-1 (Dkk1) protein is a secreted inhibitor of canonical Wnt signaling and modulates that pathway during embryonic development. It is also implicated in several diseases and hence Dkk1 is a potential target for therapeutic intervention. In the present study 6His-tagged Dkk1 expression and secretion was assessed in five mammalian cell types. Only FreeStyle 293-F cells showed significant Dkk1 protein expression in culture medium. High and stable expression of the Dkk1 protein was obtained from a selected stable FreeStyle 293-F clone 3F8, that grows in suspension in serum-free medium. The 3F8 clone showed a high Dkk1 production level (10mg/L) for up to 2 months of culture. A one step purification procedure resulting in large amounts of highly pure and active Dkk1 protein was developed. Purified Dkk1 binds its receptors LRP5 and LRP6, and is able to dose dependently inhibit canonical Wnt signaling. Recombinant Dkk1 is glycosylated, but this modification is not essential for its biological activity. In summary, an abundant source of pure and functionally active Dkk1 protein is developed that will support the identification of inhibitors such as neutralizing antibodies that could find therapeutic use.