The exact mechanisms by which probiotics confer their benefits on human health are at present only partly understood. Different modern molecular biology based technologies including comparative genomics and functional genomic analysis have been employed to unravel the mechanisms underlying the functionality of probiotics. In this context, different genes and molecules have been identified to be involved in the interactions between probiotics and the human host as well as with enteropathogens. The genes and molecules crucial for probiotic functionality are involved in survival and adaptation of probiotics to the gut niche including tolerance to the harsh conditions of the gastrointestinal tract and nutritional adaptation. The probiotics interact with enteropathogens, gut microbial symbionts and the human host by different mechanisms. Their interactions with enteropathogens and gut microbial symbionts are mediated through nutrient-based competition and synergies, competitive exclusion and production of antimicrobial compounds. Conversely, the interactions with the human host including metabolic interactions are primarily mediated through modulation of mucosal barrier functions and modulation of the innate and adaptive immune system. During recent years, our understanding of these interactions has been expanded and the present chapter discusses recent findings unravelling the functional genes and molecules of probiotics.