Newborns and children are at a much higher risk to adverse drug reactions, which is a significant public health problem. This is partially due to the lack of knowledge of the ontogeny of drug-metabolizing enzymes (DMEs). There are two types of DMEs, known as Phase-I and Phase -II DMEs. Phase I DMEs catalyze oxidation, reduction and hydrolysis reactions. Cytochrome P450s (CYPs) are a superfamily of Phase I oxidative enzymes responsible for the metabolism of a wide spectrum of drugs. Phase-II DMEs are responsible for various conjugation reactions including UDP glucuronidation, sulfonation and glutathione conjugation. Gut microbiome is known to regulate xenobiotic metabolism in adult mouse liver, but little is known of its potential involvement in the ontogeny of DMEs. The goal of this study was to determine the developmental regulation of Phase I and Phase II DMEs using germ free (GF) mice as a model. We bred both control and GF mice under the same housing conditions, and collected livers from male mice at the following ages: Days 1, 5, 10, 15, 25, 60, and 120. We isolated total RNAs and determined the RNA concentration using a Nanodrop spectrophotometer. We also determined the RNA integrity using gel electrophoresis by visualizing the 28S and 18S ribosomal RNA subunits. Using RT-qPCR we quantified the mRNAs of major DMEs and evaluated differences between control and GF mice using T-Test. As compared to age-matched control mice, the mRNAs of the xenobiotic-metabolizing Cyp3a11 was down-regulated in livers of GF mice at multiple ages including Days 10, 60, 120. Conversely, the expression of the lipid-metabolizing enzyme Cyp4a14 was up-regulated at the following ages: Days 5, 15, 60, and 120 in GF mice. In conclusion, our data suggest that the ontogeny of some DMEs is profoundly modified by lack of gut microbiota.