Delta 6 fatty acid desaturases (FADS6) have different substrate specificities that impact the ratio of omega-6/omega-3 polyunsaturated fatty acids, which are involved in regulating multiple signalling pathways associated with various diseases. For decades, FADS6 with different substrate specificities have been characterized and the functions of these crucial enzymes have been investigated, while it remains enigmatic that the substrate specificities of FADS6 from various species have a huge difference. This review summarizes the substrate specificities of FADS6 in different species and reveals the underlying relationship. Further evaluation of biochemical properties has revealed that the FADS6 prefer linoleic acid that is more hydrophilic and stable. Domain-swapping and site-directed mutagenesis have been employed to delineate the regions and sites that affect the substrate specificities of FADS6. These analyses improve our understanding of the functions of FADS6 and offer information for the discovery of novel biological resources.