Gene expression profiling of the first molar tooth germ at embryonic days (E)17.5 and 18.5, and at postnatal days (P)0, 2, and 6 from peroxisome proliferator‐activated receptor‐α (PPAR‐α) knockout mouse and from wild‐type mouse was carried out using microarrays and validated using real‐time reverse transcription–polymerase chain reaction (RT‐PCR) and western blotting. When comparing expression profiles at each time‐point, a total of 1,235 genes showed significantly different expression, 772 of which exhibited significantly decreased expression in tooth germ from knockout mouse. With genes exhibiting significantly decreased levels of expression in tooth germ from PPAR‐α knockout mouse, bioinformatic analysis using ingenuity pathway analysis yielded significant associations to cellular functions related to cellular growth/proliferation and to networks related to regulation of calcium homeostasis. Using scanning electron microscopy to investigate molars from adult PPAR‐α knockout mouse, the molar size was found to be slightly reduced, the enamel structure was found to be normal, but cervical molar enamel exhibited evidence suggesting hypomineralization. Although the PPAR‐α knockout had no significant effect on molar morphology, the results suggest that active PPAR‐α signaling is required to achieve normal mineralization of molar enamel, most probably through regulation of calcium homeostasis and metabolism of vitamin D. Cyp27b1 was expressed in tooth germ, suggesting that tooth germ can synthesize active vitamin D. Expression of Cyp27b1 was significantly enhanced in postnatal PPAR‐α knockout tooth germ.