Cerberus is a multifunctional antagonist implicated in embryonic patterning through modulation of Nodal, BMP and Wnt signals. Although its function is largely conserved in chordates, certain activities have diverged, even among vertebrates. Moreover, the antagonistic action of Cerberus from the basal chordate amphioxus toward Nodal, BMP and Wnt signals remains elusive. Here, we compared the activity of amphioxus and Xenopus Cerberus proteins using cross-species assays. We found that amphioxus and Xenopus Cerberus proteins display similar activities in antagonizing Nodal-induced events, but they exhibit both shared and distinct activities in modulating BMP and Wnt signals. Amphioxus Cerberus has reduced neuralizing activity that is dependent on inhibition of BMP signaling, and it modulates the signals of a restricted subset of Wnt proteins. Furthermore, we revealed that Xenopus Cerberus interacts with Wnt4 and Wnt11 to activate canonical Wnt signaling, whereas amphioxus Cerberus lacks this activity. These differences may be correlated with the divergence in the N-terminal region of Cerberus proteins between amphioxus and Xenopus. Our results indicate that chordate Cerberus proteins have evolved sub-functionalities that depend not only on their concentrations, but also on the properties of BMP and Wnt signals. This may account for their evolutionary distinct functions in diff erent patterning processes.