The secreted Wnt signaling inhibitor Dickkopf1 (Dkk1) plays key role in vertebrate head induction. Its receptor Kremen synergizes with Dkk1 in Wnt inhibition. Here we have carried out expression and functional studies of the Dkk and Kremen genes in amphioxus (Branchiostoma belcheri). During embryonic and larval development, BbDkk1/2/4 is expressed in the posterior mesoendoderm, anterior somatic mesoderm and the pharyngeal regions. Its expression becomes restricted to the pharyngeal region on the left side at larval stages. In 45 h larvae, BbDkk1/2/4 is expressed specifically in the cerebral vesicle. BbDkk3 was only detected at larval stages in the mid-intestine region. Seven Kremen related genes were identified in the genome of the Florida amphioxus (Branchiostoma floridae), clustered in 4 scaffolds, and are designated Kremen1-4 and Kremen-like 1-3, respectively. In B. belcheri, Kremen1 is strongly expressed in the mesoendoderm during early development and Kremen3 is expressed asymmetrically in spots in the larval pharyngeal region. In luciferase reporter assays, BbDkk1/2/4 can strongly inhibit Wnt signaling, while BbDkk3, BbKremen1 and BbKremen3 can not. No co-operative effect was observed between amphioxus Dkk1/2/4 and Kremens, suggesting that the interaction between Dkk and Kremen likely originated later during evolution.