Tamoxifen is therapeutically employed for breast and ovarian cancers, and it is also widely utilized to activate Cre recombinase in transgenic mice containing Cre-ERT locus. However, high dose tamoxifen (HDTAM) has been reported to induce many side effects in several organs and tissues. Intestinal stem cells (ISCs) play pivotal roles in sustaining the epithelial homeostasis and intestinal functionality. In this study, we systematically investigated the influences of HDTAM on ISCs and their niche. It was found that HDTAM treatment decreased the body weight and the length of small intestines (SI), damaged the gross and histological morphology of SI. Notably, HDTAM dramatically inhibited the proliferation, differentiation, gene expression of ISCs in vivo and in vitro. RNA-Seq results demonstrated that these changes caused by HDTAM were significantly correlated with the degradation of intestinal fatty acids and the process of fatty acid oxidation. Mechanistically, HDTAM impaired the morphology and function of mitochondria of intestinal epithelial cells, increased the endoplasmic reticulum (ER) contents in Paneth cells. Therefore, we concluded that HDTAM could result in a disruption for the function and homeostasis of ISCs, and the interruption of fatty acid utilization might be responsible for these effects. This study implicates a careful use and evaluation of tamoxifen is in necessity when it's used for intestinal research.