Abstract Objectives Calcium (Ca2+) is a vital micronutrient for many physiological functions with the greatest rate of accumulation occurring during the critical period of infancy. Previous work has demonstrated that molecular mechanisms of intestinal Ca2+ absorption across the small intestine are significantly different in animal models of infants and adults to permit greater absorption early in life. The colon contributes to overall Ca2+ balance in adults via transcellular, TRPV6 mediated and paracellular claudin-2 and -12 mediated pathways. Whether these same colonic pathways contribute to overall Ca2+ absorption in infants is not known. Here we aimed to investigate the molecular details of Ca2+ absorption across the large intestine in murine models of infancy relative to older mice. Methods Mice at 14 days (P14) were used as a model of suckling infants and mice at 2 months were employed to represent adult physiology. Wildtype and mutant mice with a non-functioning Trpv6 or deletions of Cldn2 or Cldn12 were used. Net 45Ca2+ flux (JCa) and Ca2+ permeability (PCa) were measured in Ussing chambers. Gene expression was determined by real-time PCR. Results JCa indicates net absorption across the colon at both P14 and 2 months. While gene expression of Trpv6 and S100g suggest greater cellular uptake of Ca2+ into colonocytes at P14, net JCa in vitro was not different than at 2 months. In contrast to previous work in mice at 2 months, TRPV6 does not mediate JCa at P14. PCa was 20% greater at P14 than 2 months, suggesting greater capacity for bidirectional diffusion of Ca2+ down an electrochemical gradient in younger mice. In contrast to previous work in mice at 2 months, claudin-2 and claudin-12 do not mediate PCa at P14 and, expression of Cldn2 and Cldn12 were significantly reduced in younger mice. Conclusions These results improve our understanding of intestinal Ca2+ handling during a critical age early in life. Future work is required to delineate molecular details under in vivo conditions of colonic Ca2+ transport in infants. Funding Sources This work was funded by grants from the Women and Children's Health Research Institute, which is supported by the Stollery Children's Hospital Foundation, and the National Sciences and Engineering Research Council to RTA, who is the Canada Research Chair in Renal Epithelial Transport Physiology.