Infant colic is a distressing condition of unknown etiology. An aberrant gastrointestinal microbiota has been associated, and Lactobacillus reuteri supplementation has been shown to reduce crying and/or fussing time ('crying time') in some infants with colic. The relationship between L. reuteri gut colonization and crying time has not been examined. We investigated the relationship between L. reuteri colonization and fecal microbiota (microbial diversity and Escherichia coli), intestinal inflammation, and crying time in infants with colic, using a subset of 65 infants from the Baby Biotics trial, which randomized healthy term infants aged <13 weeks with infant colic to receive probiotic L. reuteri DSM 17938 (1 × 108 colony forming units) or placebo daily for 28 days. We observed an overall reduction in median crying time, regardless of L. reuteri colonization status (n = 14 colonized). There were no differences in E. coli colonization rates or densities, microbial diversity or intestinal inflammation by L. reuteri colonization status. We found that L. reuteri density positively correlated with crying time, and E. coli density negatively correlated with microbial diversity. As density of L. reuteri was associated with increased crying time, L. reuteri supplementation may not be an appropriate treatment for all infants with colic.