Levels of deoxyribonuclease I (DNase I) activity in vivo have been shown to be altered by physiological and/or pathological processes. However, no information is available on the regulation of DNase I gene (DNASE1) expression in vivo or in vitro. We first mapped the transcription start sites of DNASE1 in human pancreas and in the DNase I‐producing human pancreatic cancer cell line QGP‐1, and revealed a novel site ∼ 12 kb upstream of exon 1, which was previously believed to be the single transcription‐starting exon. This initiation site marks an alternative starting exon, designated 1a. Exons 1 and 1a were used simultaneously as transcription‐starting exons in pancreas and QGP‐1 cells. Promoter assay, EMSA and chromatin immunoprecipitation analysis with QGP‐1 cells showed the promoter region of exon 1a in which the Sp1 transcription factor is specifically involved in promoter activity. This is the first to be identified as a transcription factor responsible for gene expression of vertebrate DNase I genes. Furthermore, RT‐PCR analysis indicated alternative splicing of human DNASE1 pre‐mRNA in pancreas and QGP‐1 cells. Only two transcripts among eight alternative splicing products identified can be translated to produce intact DNase I protein. These results suggest that human DNASE1 expression is regulated through the use of alternative promoter and alternative splicing.