The X-linked form of Kallmann syndrome (KS), characterized by hypogonadotropic hypogonadism and anosmia, is due to mutations in the ANOS1 gene that encodes for the extracellular matrix (ECM) protein anosmin 1. Prokineticins (PKs) exert their biological functions through the activation of the G protein-coupled receptors (GPCRs) prokineticin receptor 1 and 2 (PKR1, 2), and mutations in the PK2 and PKR2 genes are involved in the pathogenesis of KS. We have previously shown interaction between PKR2 and anosmin 1 in vitro. In the current report we present evidence of the modulation of PK2/PKR2 activity by anosmin 1, since this protein is able to enhance the activation of the ERK1/2 (extracellular signal-regulated kinase 1/2) pathway elicited by PK2 through PKR2. We also show that the N-terminal region of anosmin 1, capable of binding to the PK2-binding domain of PKR2, seems to be responsible for this effect. The whey acidic protein domain (WAP) is necessary for this modulatory activity, although data from GST pull-down (glutathione-S-transferase) and analysis of the N267K mutation in the fibronectin type III domain 1 (FnIII.1) suggest the cysteine-rich (CR) and the FnIII.1 domains could assist the WAP domain both in the binding to PKR2 and in the modulation of the activation of the receptor by PK2. Our data support the idea of a modulatory role of anosmin 1 in the biological effects controlled by the PK2/PKR2 system.