This study examines the impact of dietary fatty acids on regulation of gene expression in the mammary epithelial cells before and during puberty. The diets primarily consisted of n-9 monounsaturated fatty acids (olive oil), n-6 polyunsaturated fatty acids (safflower), saturated acids (butter), and the reference AIN-93G diet (soy oil). The dietary regimen mimics the repetitive nature of fatty acid exposure in Western diets. Dietary-induced changes in gene expression were examined in the LCM (Laser Capture Microdissected) captured mammary ductal epithelial cells at day of weaning (21 days) and at the end of puberty (50 days after birth). PCNA immunohistochemistry analysis was used to compare proliferation rates between diets. Genes differentially expressed between each of the test diets and the reference diet in both age groups were significantly enriched by cell cycle genes. Some of these genes were involved in the activation of the cell cycle pathway or the G2/M check point pathway. Cluster analysis identified an expanded set of cell cycle as well as immunity and sterol metabolism related clusters of differentially expressed genes. Fatty acid-enriched diets significantly up-regulated proliferation above the normal physiological level at day 50. The higher cellular proliferation during puberty caused by enriched fatty acid diets pose a potential increase risk of mammary cancer in later life. The human homologs of 27 of 62 cell cycle cluster of rat genes are included in a human breast cancer cluster of 45 cell cycle related genes further emphasizing the importance of our findings in the rat model.