The non-oncogene-bearing retrovirus SL3-3 murine leukemia virus induces strictly T-cell lymphomas with a mean latency of 2 to 4 months in mice of the NMRI-inbred (NMRI-i) strain. By high-throughput sequencing of retroviral tags, we have identified the genomic region carrying the transcriptional repressor and oncogene growth factor independence 1 (Gfi1) as a frequent target for SL3-3 in the NMRI-i mouse genome. Twenty-four SL3-3 insertions were identified within a 1-kb window of the 3' untranslated region (3'UTR) of the Gfi1 gene, a clustering pattern unique for this lymphoma model. Expression analysis determined that the Gfi1 gene was transcriptionally activated by SL3-3 insertions, and an upregulation of Gfi1 protein expression was detected for tumors harboring insertions in the Gfi1 3'UTR. Here we provide data in support of a mechanism by which retroviral insertions in the Gfi1 3'UTR decouple microRNA-mediated posttranscriptional regulation.