miR-20b is a member of the miR-106a-363 gene cluster, located on the X chromosome. miR-20b regulates the expression of multiple genes in vivo and it is closely related to the occurrence and development of many diseases. These diseases include inflammatory diseases and tumor development, amongst others. However, few studies have focused on the regulation of the miR-20b gene itself. In this study, we are using the miRBase database to obtain the upstream 2000 bp sequence of the miR-20b precursor. Bioinformatics software P-MATCH 1.0 and AliBaba2 werethen used to predict transcription factor binding in the upstream region. Sp-1 was identified as one of the most probable transcription factors regulating miR-20b gene expression. After treatment with a Sp-1 siRNA, the expression of miR-20b was significantly increased in RAW264.7 cells, as well as peritoneal and alveolar macrophages. In addition, the interference with Sp-1 gene expression also reversed the decrease in miR-20b expression in RAW264.7 cells induced by TNF-α. These results indicate that Sp-1 negatively regulates the expression of miR-20b in macrophages. This finding suggests the potential of Sp-1 as a target for therapies in diseases that are associated with miR-20b overexpression.