Simple SummaryCarbonic anhydrases (CAs) play a crucial role in numerous physiological and pathological processes in animals. Specifically, carbonic anhydrase VB (CA5B) is integral to both gluconeogenesis and lipogenesis. Structural variations, particularly retrotransposon insertion polymorphisms (RIPs), can contribute significantly to these processes. When an RIP is inserted into a genome region containing functional elements (such as promoters, enhancers, and insulators), it can have an impact on both the expression and phenotypic changes of target genes. In this study, we identified an RIP located in the first intron of theCA5Bgene, inserted by the SINE element, which enhanced the core promoter activity of the target gene. Through experimental analysis and growth data provided by the pig breeding farm (Anhui academy of agriculture sciences), we found that the SINE insertion improved the expression of the porcineCA5Bgene in adipose tissue. Additionally, the number of (ATTT) repeats within the SINE insertion at this site was inconsistent across breeds and individuals. This study provides insight into the role RIPs play in genetic variation ofCA5Bgenes and its phenotypic expression in pigs.AbstractTransposons are genetic elements that are present in mammalian genomes and occupy a large proportion of the pig genome, with retrotransposons being the most abundant. In a previous study, it was found that a SINE retrotransposon was inserted in the 1stintron of theCA5Bgene in pigs, and the present study aimed to investigate the SINE insertion polymorphism in this gene in different pig breeds. Polymerase chain reaction (PCR) was used to confirm the polymorphism in 11 pig breeds and wild boars), and it was found that there was moderate polymorphism information content in 9 of the breeds. Further investigation in cell experiments revealed that the 330 bp SINE insertion in the RIP-CA5Bsite promoted expression activity in the weak promoter region of this site. Additionally, an enhancer verification vector experiment showed that the 330 bp SINE sequence acted as an enhancer on the core promoter region upstream of theCA5Bgene region. The expression ofCA5Bin adipose tissue (back fat and leaf fat) in individuals with the (SINE+/+) genotype was significantly higher than those with (SINE+/−) and (SINE−/−) genotypes. The association analysis revealed that the (SINE+/+) genotype was significantly associated with a higher back fat thickness than the (SINE−/−) genotype. Moreover, it was observed that the insertion of SINE at the RIP-CA5Bsite carried ATTT repeats, and three types of (ATTT) repeats were identified among different individuals/breeds (i.e., (ATTT)4, (ATTT)6and (ATTT)9). Overall, the study provides insights into the genetic basis of adipose tissue development in pigs and highlights the role of a SINE insertion in theCA5Bgene in this process.