RING (really interesting new gene) finger protein 166, or RNF166, belongs to a C3HC4 ubiquitin ligases subfamily, which include four related proteins containing a conserved C3HC4 (Cys3-His-Cys4) RING finger domain. RNF125, one member of the subfamily, has been identified as a regulator of T cell activation, but the potential roles of another member RNF166 remains poorly understood. Here we reported that RNF166 is involved in regulation of T cell activation. Flow cytometry (FCM) data showed that overexpression of RNF166 in primary T cells and Jurkat T cells induced over 2-fold increase of CD69, a T-cell activation marker, suggesting that RNF166 is a positive-regulator of T cell activation. Furthermore, pull-down assays showed that RNF166 can bind with both Lys⁴⁸-linked polyubiquitin and Lys⁶³-linked polyubiquitin, indicating that RNF166 may play regulating roles in T cell activation by self-degradation via ubiquitin-proteasome pathway and/or cross-talk with certain signaling pathways via non-proteasome-dependent pathways. In conclusion, our work reveals that RNF166 is a potential positive-regulator of T cell activation and these findings provide a novel insight into understanding the functions of RNF166 in the positive regulation of immune responses.