Water-soluble vitamins are required as enzyme cofactors in a wide variety of metabolic reactions. Riboflavin, niacin, and vitamin C are essential in redox reactions; thiamine and biotin are involved in macronutrient metabolism; and folate, vitamin B12, pyridoxine, and riboflavin play important roles in the regulation of S-adenosylmethionine production and DNA synthesis. Each of the water-soluble vitamins appears to require its own membrane transport process for absorption across the enterocyte. The absorption of vitamin B12, or cobalamin (Cbl), is unique in requiring multiple processes from the stomach to the ileum that involve at least four different binding proteins. Whereas all water-soluble vitamins are absorbed from the small intestine, folate, biotin, and riboflavin can be transported across colonic epithelial cells, with uncertain clinical significance. This article reviews recent studies on the requirement, metabolism, and deficiency state of each water-soluble vitamin, followed by a discussion of current knowledge on the regulation of its intestinal absorption.