The human ESR2 gene codes for estrogen receptor β1 and for multiple splice variants, which are suggested to exert distinct functions in the cellular estrogen response. Given that the function of ERβ in endometrial cancer remains unclear, we examined the expression of ERβ1, ERβ2 and various further ERβ transcript variants and their association with selected cancer-related genes in 74 human endometrium samples and endometrial cancer specimens by means of RT-qPCR. Additionally, we knocked down ERβ expression in HEC-1A endometrial adenocarcinoma cells by means of siRNA transfection. Expression of four ERβ transcript variants was significantly elevated in cancer tissue or in G3 tumors compared to postmenopausal endometrium. Expression of ERβ1, ERβ2, ERβ5 and five further variants was associated with the oncogenes MYBL2 or HER2 in endometrial cancer. In addition, siRNA-triggered knockdown of ERβ expression led to a significant decline of MYBL2 mRNA and protein levels in endometrial cancer cells. Our observation of increased ERβ transcript levels in cancer tissue and particularly their correlation with the expression of oncogenes, as well as the results of our knockdown studies, suggest a role of ERβ in endometrial carcinogenesis.