TheLin28Bgene is involved in the initiation of puberty, but its regulatory mechanisms remain unclear. Therefore, in this study, we aimed to study the regulatory mechanism of theLin28Bpromoter by cloning theLin28Bproximal promoter for bioinformatic analysis. Next, a series of deletion vectors were constructed based on the bioinformatic analysis results for dual-fluorescein activity detection. The transcriptional regulation mechanism of theLin28Bpromoter region was analyzed by detecting mutations in transcription factor-binding sites and overexpression of transcription factors. The dual-luciferase assay showed that theLin28Bpromoter region −837 to −338 bp had the highest transcriptional activity, and the transcriptional activity of theLin28Btranscriptional regulatory region decreased significantly afterEgr1andSP1mutations. Overexpression of the Egr1 transcription factor significantly enhanced the transcription ofLin28B, and the results indicated thatEgr1andSP1play important roles in regulatingLin28B. These results provide a theoretical basis for further research on the transcriptional regulation of sheepLin28Bduring puberty initiation.