Endometrial cancer (EC) incidence is rising globally, creating an urgent need for novel and effective therapeutic strategies. This study aimed to investigate the anticancer effects and underlying molecular mechanisms of neferine (Nef) in EC. Through mRNA sequencing analysis, we identified the MAPK signaling pathway as a key target influenced by Nef treatment. In vitro experiments using Ishikawa cells demonstrated that Nef significantly inhibited cell proliferation, migration, and invasion abilities. Mechanistic studies revealed that Nef downregulated critical proteins in the MAPK pathway, including MEK, ERK, and JNK1, as well as metastasis-related markers such as MMP2, MMP9, and Vimentin. The MAPK activator C16-PAF was able to reverse Nef-induced suppression of MMP2 expression, further confirming the involvement of the MAPK pathway. In vivo, administration of Nef at 20 mg/kg effectively suppressed tumor growth and inhibited MAPK pathway activation in a xenograft mouse model. Compared with current therapies, which often have considerable side effects and limited efficacy especially in advanced stages, Nef’s multi-targeted mechanism of action and natural origin suggest its potential as a promising candidate for EC treatment. Future studies should focus on evaluating its therapeutic efficacy in combination with standard treatments, as well as its pharmacokinetic properties and safety profile in clinical settings.