The MAPKK Byr1 is an essential component of a Ras-dependent MAPK module required for sexual differentiation in the fission yeast, Schizosaccharomyces pombe. Here we describe the genetic and molecular characterization of a highly conserved protein, Bob1, which was identified from a two-hybrid screen for Byr1-interacting proteins. Byrl and Bobl proteins coprecipitate from S. pombe cell lysates, and both proteins localize to the tips and septa of S. pombe cells. S. pombe bob1 null (bob1delta) mutants lack obvious growth defects but exhibit a significant mating deficiency, which can be suppressed by overexpression of Byrl. Overexpression of Bob1 also leads to inhibition of mating in S. pombe, and this defect is likewise suppressed by Byrl overexpression. Bob1 is highly homologous in structure to the mammalian MM-1/Pfd5 and budding yeast Gim5/Pfd5-Sc proteins, which have been implicated as regulators of actin and tubulins. Similar to budding yeast gim5/pfd5-Sc mutants, S. pombe bob1delta cells have cytoskeletal defects, as judged by hypersensitivity to cytoskeletal disrupting drugs. byr1delta mutants do not share this characteristic with bob1delta mutants, and byr1delta bob1delta mutants are not significantly more sensitive to cytoskeletal disrupting drugs than cells carrying only the bob1delta mutation. Taken together, our results suggest that Bob1 has Byr1-related function(s) required for proper mating response of S. pombe cells and Byrl-independent function(s) required for normal cytoskeletal control. We show that the human MM-1/Pfd5 protein can substitute for its counterpart in fission yeast, providing evidence that the functions of Bob1-related proteins have been highly conserved through evolution. Our results lead us to propose that Bob1-related proteins may play diverse roles in eukaryotic organisms.