The gene luxS is highly conserved among members of the family Pasteurellaceae, including the bovine pathogen Histophilus somni. LuxS has been characterized as an S-ribosylhomocysteine lyase and contributes to methionine metabolism. In many bacteria, LuxS is also involved in synthesis of autoinducer-2 (AI-2), which contributes to quorum sensing and biofilm formation. We determined that culture supernatants from different members of the Pasteurellaceae differ substantially in their ability to induce luminescence by the Vibrio harveyi AI-2 mutant strain BB170. However, culture supernatant from H. somni did not induce any luminescence in strain BB170, but luxS from H. somni did complement AI-2 production and induce luminescence by BB170 from culture supernatant of Escherichia coli DH5-alpha, which has a luxS frameshift mutation. Therefore, luxS in H. somni is functional. H. somni is also capable of forming a prominent biofilm, but a transposon mutation in luxS in strain 2336 (2336::TnluxS) was not noticeably deficient in biofilm formation, but was highly attenuated in mice and bovines. The electrophoretic profile of the lipooligosaccharide and the intracellular survival and trafficking of the bacterium post-phagocytosis were substantially altered compared to the parent strain, but exopolysaccharide production was not affected. Furthermore, 2336::TnluxS became elongated after co-incubation with monocytes, but not monocyte-free culture medium. To clarify whether LuxS contributes to gene regulation, mRNA from the parent strain and 2336::TnluxS was sequenced from bacteria growing planktonically and in a biofilm. RNA-sequencing indicated that mutation of luxS down-regulated many genes involved in metabolic functions in H. somni, particularly in purine and pyrimidine metabolism, pyruvate, methionine, and sulfur metabolism, and in lysine and pantothenate and CoA biosynthesis. The broad nature of the genes affected indicated that LuxS in H. somni contributes to regulatory functions that affect virulence, but does not contribute to quorum sensing or biofilm formation.