Moloney murine leukemia virus (M-MLV) replication is restricted in embryonic carcinoma (EC) and embryonic stem (ES) cells, likely to protect the germ line from insertional mutagenesis. Proviral DNAs are potently silenced at the level of transcription in these cells. This silencing is largely due to an unidentified trans-acting factor that is thought to bind to the primer binding site (PBS) of M-MLV and repress transcription from the viral promoter. We have partially purified a large PBS-mediated silencing complex and identified TRIM28 (Kap-1), a known transcriptional silencer, as an integral component of the complex. We show that RNAi-mediated knockdown of TRIM28 in EC and ES cells relieves the restriction and that TRIM28 is bound to the PBS in vivo when restriction takes place. The identification of TRIM28 as a retroviral silencer adds to the growing body of evidence that many TRIM family proteins are involved in retroviral restriction.