Tamoxifen (TAM) is a selective estrogen receptor modulator (SERM) widely used for adjuvant therapy of breast cancer. An important side-effect of TAM is increased risk of endometrial cancer. To investigate mechanisms underlying this effect we examined transcriptional and epigenetic changes in human and monkey uterus, normal human mammary epithelial cells (NHMECs), and human endometrial stromal cells (HESCs). Uterine DNA from women (n = 9), Erythrocebus patas monkeys (n = 3), and Macaca fascicularis monkeys (n = 6), all exposed to TAM, showed no difference in 5-methyl-cytosine (5-meC) levels compared to unexposed controls (n = 6, 2, and 6, respectively). Microarray comparison of TAM-exposed and unexposed NHMECs and HESCs revealed cell-specific differences, with confirmation by RT-PCR. TAM-exposed NHMECs showed up-regulation of interferon signaling and immune response pathways, while TAM-exposed HESCs showed up-regulation of steroid and fatty acid biosynthesis pathways. Promoter region CpG islands for genes highly up-regulated by TAM in NHMECs (MX1 and STAT1) and in HESCs (PPARG, SREBF2, HMCGS and Prune2), did not show significant differences for 5-meC. We did observe a significant depletion of total histone H3, and dimethyl histone H3 lysines K4, K27 and K36, by Western blot, in TAM-exposed HESCs, compared to unexposed controls. Whereas TAM exposure had no discernible effect on 5-meC levels in primate uterus, TAM induced up-regulation of different transcriptional pathways in NHMECs and HESCs, and concomitantly depleted some H3 dimethyl histone lysine levels in HESCs. These findings highlight several features of transcriptional dysregulation by TAM that should be further investigated in the context of TAM-induced endometrial carcinogenesis.