S100A12 is involved in the inflammatory response and is considered an important marker for many inflammatory diseases in humans. Our previous studies indicated that the S100A12 gene was abundant in the immune tissues of pigs and was significantly upregulated during infection with Haemophilus parasuis (HPS) or porcine circovirus type 2 (PCV2). In this study, the mechanism of transcriptional regulation of S100A12 was investigated in pigs. Our results showed that S100A12, CCAAT/enhancer-binding protein beta (C/EBP beta) and activator protein-1 (AP-1) genes were up-regulated in PK-15 (ATCC, CCL-33) cells when treated with LPS or Poly I: C. Additionally, the promoter activity and expression level of the S100A12 gene were significantly upregulated when C/EBP beta or AP-1 were overexpressed. We utilized electromobility shift assays (EMSA) to confirm that C/EBP beta and AP-1 could directly bind the S100A12 gene promoter. We also found that the transcriptional activity and expression levels of C/EBP beta and AP-1 could positively regulate each other. Furthermore, the promoter activity of the S100A12 gene was higher when C/EBP beta and AP-1 were cotransfected than when they were transfected individually. We concluded that the S100A12 gene was cooperatively and positively regulated by C/EBP beta and AP-1 in pigs. Our study offers new insight into the transcriptional regulation of the S100A12 gene.