The results of our previous study suggested that in addition to the small intestinal lactase activity and transit time, colonic processing of lactose may play a role in lactose intolerance. We investigated whether colonic fermentation of lactose is correlated with lactose intolerance. After 28 Chinese subjects had undergone 1 glucose (placebo) and 2 lactose challenges, consistent lactose tolerant (n = 7) and intolerant (n = 5) subjects with no complaints after glucose administration were classified on the basis of the 6-h symptom scores. Before the challenges, fecal samples were collected for in vitro incubation with lactose. The incubation was carried out in a static system under anaerobic conditions for 5 h during which samples were taken for measurement of short-chain fatty acids, lactate, lactose, glucose, and galactose. Fecal bacterial composition was determined by fluorescent in situ hybridization. The tolerant and intolerant groups did not differ in the rate or degree of hydrolysis of lactose or production of glucose and galactose. The intolerant group produced d- and l-lactate, acetate, propionate, and butyrate significantly faster than the tolerant group. In the intolerant group, the amounts of acetate, propionate, butyrate, and l-lactate produced were higher than those in the tolerant group. Fecal bacterial composition did not differ between the 2 groups. The results indicate that the degree and rate of lactose hydrolysis in the colon do not play a role in lactose intolerance. However, after lactose is hydrolyzed, a faster and higher production of microbial intermediate and end metabolites may be related to the occurrence of symptoms.