Sirtuin-1 (SIRT1) is a multispecific lysine deacetylase which recognises and deacetylates a range of important protein substrates (such as p53, RelA and histones), thereby regulating the substrates' activity, and in turn, the cell's activity. Cellular consequences of SIRT1 activity include regulation of cell senescence, the cell cycle, gene expression and response to metabolic state. Indeed, activators of SIRT1 have been shown to increase the mean lifespan of mice, Drosophila, and S. cerevisieae. Interestingly, recent work has shown that mutating certain positions in human SIRT1 to the amino acid identities found in S. cerevisieae decreases activity against modern substrates such as p53, and increases activity against ancient substrates such as histones. This indicates that SIRT1 homologs from different organisms have different selectivity profiles. However, the evolution of these different selectivity profiles from the common ancestor of Metazoa and Fungi remain unknown. Here, we performed phylogenetic inference followed by ancestral sequence reconstruction to ressurect the evolutionary trajectory of SIRT1 from the common ancestor of Metazoa and Fungi to the present human SIRT1 and its S. cerevisieae homolog. We queried the posterior probability distributions of resurrected ancestors at key specificity-determining residue positions (SDPs) to determine the most likely amino acid identity at those positions. Our analysis indicates that there have been significant changes in these SDPs over the course of evolution. This work lays the foundation for structural and functional characterisation of ancestral configurations of these SDPs that will permit us to correlate changes in residue identity during evolution with changes in function/selectivity profile. This will undoubtedly enhance our understanding of how SIRT1 recognises its substrates, and more generally, how multispecific proteins evolve to change their selectivity profiles.