Abstract: Objective To investigate the binding proteins of PIH1D1. Methods A PIHID1 stable-expressed HEK293T cell strain was constructed using a FLAG-HA double-tagged PIHID1 expressing plasmid. After performing the FLAG-HA tandem affinity purification (TAP), the mass spectrometric was carried out to analyze the interaction protein complex of PIH1D. Results The FLAG-HA double-tagged PIHID stable-expressed HEK293T cell strain was obtained. We found a number of potential binding proteins of PIHID1 by means of the mass spectrometric, including RPAP3, UXT, PFD2 and PFD6 which were involved in RNAP II assemble factory and MONAD/WDR92 which was able to promote apoptosis. Besides we found PIP and CALM1 belonging to calmodulin-Ca2+ complex, and PKM and LCN1 which were classic enzyme of metabolism as binding proteins of PIHID1 for the first time. Conclusions PIH1D1 might participate in multiple biological processes including apoptosis, calmodulin-Ca(2+) signaling pathway, RNA polymerase II assembling, and metabolism regulation through interaction with a RPAP3，UXT，PFD2，PFD6，MONAD/WDR92，PIP，CALM1，PKM and LCN1.

Key words: Key words: PIH1D1, TAP, mass spectrometric