Vitamin A deficiency (VAD) is an overwhelming public health problem that affects hundreds of millions of people worldwide. A definitive solution to VAD has yet to be identified. Because it is an essential nutrient, vitamin A or its carotenoid precursor β-carotene can only be obtained from food or supplements. In this study, we wanted to establish whether β-carotene produced in the mouse intestine by bacteria synthesizing the provitamin A carotenoid could be delivered to various tissues within the body. To achieve this, we took advantage of the Escherichia coli MG1655*, an intestine-adapted spontaneous mutant of E. coli MG1655, and the plasmid pAC-BETA, containing the genes coding for the 4 key enzymes of the β-carotene biosynthetic pathway (geranylgeranyl pyrophosphate synthase, phytoene synthase, phytoene desaturase, and lycopene cyclase) from Erwinia herbicola. We engineered the E. coli MG1655* to produce β-carotene during transformation with pAC-BETA (MG1655*-βC) and gavaged wild-type and knockout mice for the enzyme β-carotene 15,15′-oxygenase with this recombinant strain. Various regimens of bacteria administration were tested (single vs. multiple and low vs. high doses). β-Carotene concentration was measured by HPLC in mouse serum, liver, intestine, and feces. Enumeration of MG1655*-βC cells in the feces was performed to assess efficiency of intestinal colonization. We demonstrated in vivo that probiotic bacteria could be used to deliver vitamin A to the tissues of a mammalian host. These results have the potential to pave the road for future investigations aimed at identifying alternative, novel approaches to treat VAD. J. Nutr. 144: 608-613, 2014.