SUMMARY Understanding the evolution of protein function is a central goal in evolutionary biochemistry. Most mechanistic work on this problem has focused on single proteins with single functions; however, proteins often work as members of multi-functional, multi-protein complexes. How can evolution optimize one protein function without breaking another? How are new proteins integrated into multi-protein complexes? Answering these and other questions requires mechanistic studies of the natural evolution of multi-functional, multi-protein complexes. To answer these questions, we propose studying the evolution of Toll-like receptor 4 (TLR4)—a multi-protein, multi-functional protein complex found in vertebrate animals. This complex is central to how animals respond to both infection and tissue damage. It is the subject of intense interest, both from the perspective a basic biology and as a drug target for inflammatory disorders. The complex evolved in serial over hundreds of millions of years. In this work, we will pose—and answer—three questions: 1) How did the ability of the complex to recognize pro- inflammatory signals change over the last 300 million years? 2) What were the evolutionary forces and processes that led to these functional changes? 3) How did a completely new protein evolve in the ancestor of land animals and become integrated into the complex? To answer these questions, we will employ a combination of phylogenetic analysis, ancestral sequence reconstruction, high-throughput protein characterization, biochemical/biophysical studies, careful functional characterization, and in vivo experiments in zebrafish. This work will provide unprecedented understanding of the mechanisms—both biochemical and evolutionary— that lead to new function in multi-functional protein complexes. By specifically studying the evolutionary process that led to the human TLR4 complex, this work will also provide a lens through which we can interpret animal model studies of innate immunity and apply their findings to human biology. And, finally, an evolutionary approach is a powerful means to dissect how proteins work. Our results will provide deep insight into how this important protein complex functions and can be manipulated to achieve better human health outcomes.