Introduction Stem cells mediate cyclic regeneration of the endometrium. The upregulated expression of receptors and modulators of the notch signaling pathway in endometriosis suggests an involvement in the pathogenetic process. Here, we investigated the effects of notch pathway inhibition by a γ‐secretase inhibitor (GSI) on stemness‐associated properties of the epithelial endometriotic cell line 12Z and of primary endometriotic stroma cells. Material and methods 12Z cells and primary endometriotic stroma cells of 7 patients were treated with or without GSI, and analyzed for changes in gene expression by TaqMan low‐density arrays, quantitative PCR, and flow cytometry. The functional impact of GSI treatment was studied by MTT assay, cell cycle analysis, colony formation assay, annexin V apoptosis assay, and aldehyde dehydrogenase activity assays. Results In 12Z cells, GSI treatment reduced aldehyde dehydrogenase activity and colony formation, and induced a shift to the G2/M phase of the cell cycle. Cell viability was decreased and apoptosis was increased in both cell models. GSI further induced transcriptional downregulation of the stemness‐associated factors leukemia inhibitory factor receptor (LIFR), sex‐determining region Y (SRY)‐ box 2, interferon‐induced transmembrane protein 1, and hes‐related family bHLH transcription factor with YRPW motif 1, in 12Z cells and in primary cell cultures. Downregulation of LIFR expression by GSI was confirmed at the protein level by flow cytometry. Conclusions Our in vitro data suggest that application of GSI may be a worthwhile approach in the treatment of endometriosis that warrants further investigation.