Mammalian milk/colostrum contains from trace to ∼13% of carbohydrate, of which lactose (Gal(β1-4)Glc) usually constitutes more than 80%. Milk/colostrum of most mammals also contains a variety of oligosaccharides, many of which have N-acetylglucosamine, galactose, fucose, and/or sialic acid residues attached to lactose, which is always located at the reducing end. The ratio of milk oligosaccharides to free lactose in milk/colostrum varies, depending on the mammalian species. Most human milk oligosaccharides (HMOs) cannot be digested within the small intestine, and reach the colon where they stimulate the growth of bifidobacteria and act as possible antiadhesion factors against pathogenic microorganisms in the human infant. The biological functions of milk oligosaccharides have recently been an active field of research. In this article, we describe the chemical structures and methods for the structural analysis of milk oligosaccharides, quantitative aspects and methods for their quantification, their gastrointestinal digestion and biosynthesis, and their biological significance as prebiotics, anti-infection factors, receptor analogues, and immunomodulators, with special reference to the oligosaccharides of human milk. We also describe the milk oligosaccharides of domestic farm animals and discuss comparative aspects of milk oligosaccharides among mammalian species, as well as future trends and the industrial utilization of milk oligosaccharides.