Objective The spermatogenesis- and oogenesis-specific bHLH transcription factor-1 (SOHLH1) andSRY box (SOX30) are transcription factors related to spermatogenesis. Sohlh1 and Sox30 knockout male mice lose fertility.At 7 days after birth, the Sohlh1 knockout male mouse DNA chip exhibits significant down-regulation of Sox30 expression.This study explored the role of the early development-related transcription factor SOHLH1 in the transcriptional regulation ofthe key gene Sox30 during late spermatogenesis. Methods The Sox30 promoter luciferase expression plasmid wasconstructed, and then together with the Sohlh1 eukaryotic expression plasmid was transiently co-transfected. Thefluorescence value was then detected by a luciferase activity assay. The primary activation site was screened using achromatin immunoprecipitation assay to detect the binding of SOHLH1 to the Sox30 promoter DNA sequence. Results Theluciferase activity was higher in the experimental group co-transfected with the Sohlh1 eukaryotic expression plasmid and theSox30 promoter region luciferase plasmid than in the control group. SOHLH1 binding to specific sites was strongest on theSox30 promoter binding sites at -489 bp. Conclusions The early important transcription factor SOHLH1 can directlyactivate acrosome-forming gene Sox30 transcription, with -489 bp (CAGGTG) as the main specific binding site. Thisaction enriches the regulation of delayed translation expression during spermatogenesis. The study thus provides a preliminary exploration of the regulatory mechanism.