Long-chain acyl-CoA synthetase 1 (ACSL1) plays an important role in fatty acid metabolism and fat deposition. The transcription of the gene is regulated specifically among cells and physiological processes, and transcriptional regulation of in adipogenesis remains elusive. Here, we characterize transcription factors (TFs) associated with adipogenesis in the porcine gene. CCAAT-enhancer binding protein (C/EBP)α, a well-known adipogenic marker, was found to enhance the expression of the gene via binding two tandem motifs in the promoter. Further, we demonstrate that ACSL1 mediates C/EBPα effects on adipogenesis in preadipocytes cultured from subcutaneous fat tissue of pigs via gain- and loss-of-function analyses. The cAMP-response element binding protein, another TF involved in adipogenesis, was also identified in the regulation of gene expression. Additionally, single nucleotide polymorphisms (SNPs) were screened in the promoter of among four breeds including the Chinese indigenous Min, and Duroc, Berkshire, and Yorkshire pigs through sequencing of PCR products. Two tightly linked SNPs, -517G>T and -311T>G, were found exclusively in Min pigs. The haplotype mutation decreases promoter activity in PK-15 and ST cells, and in vivo the expression of , illustrating a possible role in adipogenesis regulated by C/EBPα/ACSL1 axis. Additionally, a total of 24 alternative splicing transcripts were identified, indicating the complexity of alternative splicing in the gene. The results will contribute to further revealing the regulatory mechanisms of during adipogenesis and to the characterization of molecular markers for selection of fat deposition in pigs.