Syntenin and Pick1 (PDZ-domain proteins) have been reported to bind to ephrinB ligands. However, there is no data related to whether ephrinB ligands, located on the membrance of osteoclasts, regulate expression levels of syntenin and Pick1, following activation by EphB receptors. RAW264.7 cell line was used as osteoclast precursors, which can differentiate into osteoclast induced by RANKL. Western blot analysis and/or immunofluorescence staining revealed that not only syntenin and Pick1, but also ephrinB2 were prominently expressed during RANKL-induced osteoclast differentiation of RAW264.7 cells, thus furtherly illustrating that the model of RANKL-induced osteoclast differentiation of RAW264.7 cells can be used for further investigation. In order to study the effects of reverse signaling on the expression levels of PDZ-domain proteins, soluble EphB4-Fc protein was used to stimulate ephrinB2 because EphB4 exclusively interacts with ephrinB2. In contrast to the similar expression level of syntenin between EphB4-Fc and Fc treated group, the protein and mRNA expression levels of Pick1 were obviously enhanced in EphB4-Fc treated group compared with Fc treated group. However, co-immunoprecipitation results showed that there were no direct interactions between ephrinB2 and endogenously expressed syntenin and Pick1 in the RANKL-induced osteoclasts of RAW264.7 cells in vitro. In summary, it was demonstrated that both syntenin and Pick1 were expressed during RANKL-induced osteoclast differentiation of RAW264.7 cells, and EphB4/ephrinB2 reverse signaling regulates the expression levels of Pick1, but not of syntenin. These data help to preliminarily explore the potential PDZ-domain proteins involved in the downstream of ephrinB2 during the osteoclast differentiation of RAW264.7 cells in vitro.