Endometrial cancer (EC) is one of the most common tumors in the female reproductive system, with usually limited curative effect and adverse reactions. Neferine (Nef) is derived from lotus seeds, a medicinal and edible plant, and has good functional activity. However, its efficacy and anti-cancer mechanism for EC remain to be studied. In this study, MTT was used to test cytotoxicity, and flow cytometry was used to detect cell cycle, apoptosis, Ca²⁺ levels, and mitochondrial membrane potential (MMP) changes. miRNA-seq detected differentially expressed miRNAs after Nef treatment. In addition, WB and immunohistochemistry were used to detect apoptosis-related proteins in cells and mice.Our results showed that Nef can cause ishikawa cell apoptosis and block cell proliferation through the G2/M phase. We obtained 101 significantly different miRNAs (p < 0.05 and |logFC| > 1) through miRNA sequencing, conducted GO and KEGG enrichment analysis, and obtained the Ca²⁺ and PI3K/AKT signaling pathways that are highly related to apoptosis. Subsequently, we found that Nef treatment significantly changed intracellular Ca²⁺ levels and MMP, activating the endoplasmic reticulum stress (ERS) pathway and the expression of key proteins in the mitochondrial pathway. In addition, Nef also inhibited the expression of key proteins in the PI3K/AKT pathway, causing cell apoptosis. Moreover, in mouse tumor tissues, the expression of key proteins such as CHOP, Bcl-2, Caspase 3, Cyto-c, and p-AKT was also consistent with the results in vitro. In conclusion, Nef can induce the activation of the mitochondrial pathway through the Ca²⁺-mediated ERS pathway and PI3k/AKT pathway, thereby inducing apoptosis in endometrial cancer cells, confirming the potential function of Nef in the prevention and treatment of EC.