The aim of this study is to investigate the potential inhibitory effect of alpha-chaconine and alpha-solanine on RL95-2 estrogen receptor (ER) positive human endometrial cancer cell line and to identify the effect of these glycoalkaloids on the Akt signaling and ER alpha. The cell proliferation profiles and the cytotoxicity studies were performed by Real-Time Cell Analyzer (xCELLigence) and compared with Sulphorhodamine B (SRB) assay. The effects of alpha-chaconine (2.5, 5, 10 mu M), alpha-solanine (20, 30, 50 mu M), API-1 (25 mu M) and MPP (20 mu M) effects on Akt (Ser473) and ER alpha (Ser167) expressions evaluated by Western blot and qPCR method. Their IC50 values were as alpha-chaconine (4.72 mu M) < MPP (20.01 mu M) < alpha-solanine (26.27 mu M) < API-1 (56.67 mu M). 10 mu M alpha-chaconine and 20, 30 and 50 mu M alpha-solanine were effective in decreasing p-Akt(Ser473)/Akt ratio compared to positive control API-1. When the p-ER alpha/ER alpha ratios were evaluated, it was observed that alpha-chaconine (2.5, 5, 10 mu M) and alpha-solanine (50 mu M) were as effective as the specific ER alpha inhibitor MPP in reducing the ratio of p-ER alpha/ER alpha compared to the control group. In conclusion, it has been shown that the proliferation of alpha-chaconine and alpha-solanine in human endometrial carcinoma cells reduces the expression and activity of the Akt and ER alpha signaling pathway.