Ubiquitination plays very important roles in regulating protein degradation and transportation. Many RING finger proteins act as E3 ligase in these processes. A new typeⅠ transmembrane protein with unknown function, named RNF148 was studied. A RFP -IRES- TSPANs-EGFP/pCI vector was constructed to screen the substrate of RNF148. By using FACS, co-IP and confocal test, RNF148 was identified to interact with TSPAN15 which belongs to a tetraspanin family. RNF148 could down-regulate TSPAN15 by its E3 ligase activity. The RING domain of RNF148 was a key structure of its ubiquitin-ligase activity and was required for the poly-ubiquitination of TSPAN15. The mutation of Lys-21 and Lys-278 to arginine in the cytosolic fragment of TSPAN15 led to low ubiquitination density compared with the wild-type. RNF148-mediated ubiquitination promoted proteasomal degradation and downregulation of TSPAN15 via Lys-29 linkages. RNF148 also ubiquitinated TSPAN15 via generating Lys-63 polymerize ubiquitin molecules. These findings demonstrate for the first time that RNF148 is a RING finger ubiquitin E3 ligase and TSPAN15 is one of its substrates.