Simple SummaryThe low-density lipoprotein receptor (LDLR) family plays crucial roles in lipid metabolism, but this family has not been investigated in ancient chordates (protochordates) to date. We identified and characterized a new LDLR family member in a protochordate—Branchiostoma belcheri. Additionally, we investigated the evolutionary process and molecular function in this gene family. Our work fills a research gap in the LDLR family in protochordates and provides new insights into the origin and evolution of chordate LDLRs.AbstractLow-density lipoprotein receptors (LDLRs) are a class of cell-surface endocytosis receptors that are mainly involved in cholesterol homeostasis and cellular signal transduction. Very-low-density lipoprotein receptors (VLDLRs), which are members of the LDLR family, have been regarded as multi-function receptors that fulfill diverse physiological functions. However, noVLDLRgene has been identified in protochordates to date. As a representative protochordate species, amphioxi are the best available example of vertebrate ancestors. Identifying and characterizing theVLDLRgene in amphioxi has high importance for exploring the evolutionary process of the LDLR family. With this study, a new amphioxusVLDLRgene (designatedAmphiVLDLR) was cloned and characterized using RACE-PCR. The 3217 nt transcript of theAmphiVLDLRhad a 2577 nt ORF, and the deduced 858 amino acids were highly conserved within vertebrate VLDLRs according to their primary structure and three-dimensional structure, both of which contained five characteristic domains. In contrast to other vertebrate VLDLRs, which had a conserved genomic structure organization with 19 exons and 18 introns, theAmphiVLDLRhad 13 exons and 12 introns. The results of a selective pressure analysis showed that theAmphiVLDLRhad numerous positive selection sites. Furthermore, the tissue expression ofAmphiVLDLRusing RT-qPCR showed thatAmphiVLDLRRNA expression levels were highest in the gills and muscles, moderate in the hepatic cecum and gonads, and lowest in the intestines. The results of the evolutionary analysis demonstrated that theAmphiVLDLRgene is a new member of the VLDLR family whose family members have experienced duplications and deletions over the evolutionary process. These results imply that the functions of LDLR family members have also undergone differentiation. In summary, we found a newVLDLRgene homolog (AmphiVLDLR) in amphioxi. Our results provide insight into the function and evolution of theLDLRgene family.