Objective：To study the binding of exogenous tumor necrosis factor receptor-associated factor 6(TRAF6)to Krüppel-like factors 5(KLF5)as well as the pattern and site of KLF5 polyubiquitination by TRAF6 in HEK 293T(i.e. 293 T)cells. Methods：The 293T cells were co-transfected with Flag-TRAF6，HA-KLF5 and ubiquitin(Ub)expression plasmids，or shTRAF6 and TRAF6 C70A plasmids in different combinations for 48 h. Then，the binding of TRAF6 to KLF5 and KLF5 K48/K63-linked polyubiquitination by TRAF6 were detected using immunoprecipitation(IP)and immunoblotting(IB)assays. Moreover，the plasmids with all lysine mutation of KLF5 were constructed，and co-transfected with TRAF6 overexpression plasmids into 293T cells. Thereafter，the level of KLF5 K63- linked polyubiquitination and the lysine(site)of KLF5 K63 -linked polyubiquitination were measured or identified by IP and IB. Results：TRAF6 and KLF5 in 293T cells could bind with each other. The overexpression of TRAF6 up - regulated while the knockdown or activity deficiency of TRAF6 down-regulated the level of KLF5 K63-linked polyubiquitination. The site of KLF5 K63- linked polyubiquitination was its K99 or K100 lysine. Conclusion：TRAF6 can interact with KLF5 and modify the K99 and K100 of KLF5 via K63-linked polyubiquitination.