TSPO was discovered in 1977 and it’s function is still currently unknown. Significant research has suggested that TSPO functions in steroidogenesis to import cholesterol from the mitochondrial outer membrane (MOM) to the mitochondrial inner membrane (MIM) where it is converted into steroids. There were two indications that this is TSPOs main function: its elevated levels in steroidogenic tissue and its primary location in the MOM. There is evidence of TSPO binding cholesterol with high affinity, however there is not currently evidence of TSPO transporting cholesterol. STAR, ACBD1, and ACBD3 are proteins thought to be associated with TSPO and steroidogenesis. However, the distribution of these proteins in various eukaryotes show little similarity suggesting that TSPO functions independently. The function of TSPO in steroid synthesis has been called into question because a well-cited research paper claimed that TSPO knockdown resulted in embryonic lethal mice, however there was no evidence presented from their study and this experiment did not produce the same results when repeated in later studies. There are also studies that show TSPO may not be involved in regulation of sterols, but instead may regulate cell stress. The elevated levels of TSPO during inflammation suggest a role for TSPO in cellular stress. Binding interactions with porphyrins and heme also support that TSPO may modulate stress levels. We used the phylogeny of TSPO in order to gain greater insight into the evolutionary function of TSPO. NCBI BLAST searches revealed that TSPO was present in bacteria and had a widespread but patchy distribution in a small set of eukaryotes. From these initial results, we were prompted to search a larger set of eukaryotes for TSPO. All of the prokaryotic and eukaryotic TSPO sequences were used to create a phylogenetic tree that would provide greater insight into the evolution and function of TSPO. If TSPO was from a common ancestor, it is probable that its function is related to sterol regulation whereas if gained in eukaryotes by horizontal gene transfer from bacteria its function is related to stress regulation. The phylogenetic tree was most consistent with an ancestral origin of TSPO with an evolutionary function related to steroid synthesis regulation. However, there is not sufficient research to confirm the function of TSPO.