Natural killer (NK) cell immunotherapy is a significant category in tumor therapy due to its potent tumor-killing and immunomodulatory effects. This research delves into exploring the mechanisms underlying the ability of amoxicillin to boost NK cell cytotoxicity in NK cell immunotherapy. Amoxicillin significantly enhances the cytotoxic activity of NK-92MI cells against MCF-7 cells by triggering the initiation of a cytolytic program in target cell-deficient NK-92MI cells and augmenting the degranulation level of NK-92MI cells in the presence of target cells. The ability of NK cells to recognize target cells was increased upon exposure to amoxicillin at low concentration (10 ng/mL). Additionally, the utilization of amoxicillin loaded in liposome (AMO@Liposome) for NK cell immunotherapy in a mouse breast cancer model resulted in an increased antitumor effect in comparison to without the treatment of AMO@Liposome. RNA transcriptome analysis showed that amoxicillin upregulated differential genes related to the synaptic vesicle cycle pathway and calcium signaling pathway, and FOSB, TNFRSF18, and H4C1 were identified as critical players. These studies suggest that the strategy of using amoxicillin in NK cell immunotherapy has potential applications in the field of tumor therapy.