The Serine Protease Inhibitor (serpin) protein has been suggested to play a key role in the interaction of bifidobacteria with the host. By inhibiting intestinal serine proteases, it might allow bifidobacteria to reside in specific gut niches. In inflammatory diseases where serine proteases contribute to the innate defense mechanism of the host, serpin may dampen the damaging effects of inflammation. In view of the beneficial roles of this protein, it is important to understand how its production is regulated. Here we demonstrate that Bifidobacterium longum NCC 2705 serpin production is tightly regulated by carbohydrates. Galactose and fructose increase the production of this protein while glucose prevents it, suggesting the involvement of catabolite repression. We identified that di- and oligosaccharides containing galactose (GOS) and fructose (FOS) moieties, including the human milk oligosaccharide Lacto-N-tetraose (LNT), are able to activate serpin production. Moreover, we show that the carbohydrate mediated regulation is conserved within B. longum subsp. longum strains but not in other bifidobacterial taxons harboring the serpin coding gene, highlighting that the serpin regulation circuits are not only species- but also subspecies- specific. Our work demonstrates that environmental conditions can modulate expression of an important effector molecule of B. longum, having potential important implications for probiotic manufacturing and supporting the postulated role of serpin in the ability of bifidobacteria to colonize the intestinal tract.