Abstract: Nowadays, fatty liver and visceral excessive lipid accumulation are common in cultured fish, which reduces survival rate, growth performance and disease resistance. Yellow catfish Pelteobagrus fulvidraco is widely distributed in rivers, lakes and other fresh waters in China, and cultured in China and several Asian countries. The fish has relatively high economic value because of delicious taste and abundant nutrition. However, due to intensive culture and improper feeding, excessive fat accumulation in abdominal cavity and liver commonly occurs, which seriously affects the taste and health of yellow catfish. Therefore, the research on fat metabolism of yellow catfish has always been the focus. Lysosomal acid lipase (LAL), encoded by LIPA, hydrolyzes cholesterylesters (CEs) and triglycerides (TGs) to cholesterol and free fatty acids (FFAs), which are then used for metabolic purposes in the cells. The studies have been conducted to explore lal structure in mammals, but were scarce in fish. For this reason, it is important to study the molecular characteristics of lal in the regulation of lipid metabolism of yellow catfish. In this study, we analyzed molecular structure, tissue expression, promoter structure and function, and transcriptional regulation of lal. The lal gene was amplified from yellow catfish by RT-PCR and RACE approaches. The cDNAs of lal was 1802 bp, encoding a peptide of 398 amino acid residues, and 5′ upstream promoter was 2052 bp in length. The molecular weight of the theoretical protein was 45.42 kDa, the isoelectric point was 7.70, and it had a signal peptide with 23 residues, five glycosylation sites, three cysteines, a catalytic ternary and a “cap” domain and a “lid” region. The amino acid alignment and phylogenetic analysis revealed that lal of P. fulvidraco was closely related to that of Ictalurus punctatus. The lal mRNA was expressed in all tested tissues (heart, liver, brain, spleen, kidney, muscle, fat, intestine, testis and ovary), with the highest expression levels in spleen, intestine and testis. Promoter sequence analysis revealed several transcription factor binding sites in lal promoter, such as Sp1, STAT3, PPARα, FOXO1, PPARγ, and HNF4α. Studies on promoter activity showed that –1507/–1016 region negatively regulated promoter activity, while –1016/+51 region positively regulated promoter activity. The present study indicated that lal mRNA was expressed in multiple high metabolism tissues. The transcription of lal was regulated by multiple transcription factors. This study help us to understand the structure and function of lal, and lay a foundation for further research on the regulatory mechanism of lipid metabolism in fish.