Background Natural killer (NK) cells are promising candidates for cancer immunotherapy due to their safety and potent anti-tumor activity. However, their therapeutic efficacy is often limited by poor persistence and activity within the tumor microenvironment (TME) caused by a lack of essential cytokines. Methods To overcome cytokine dependence, we engineered NK92, primary NK (pNK), and chimeric antigen receptor (CAR)-NK92 cells to express an interleukin-7 receptor with an insertion mutation (IL-7R-IM), which induces constitutive signaling. We evaluated the proliferation, viability, and cytotoxicity of these cells in vitro and analyzed downstream signaling pathways using RNA sequencing and western blotting. The in vivo anti-tumor efficacy was assessed using a metastatic leukemia xenograft mouse model. Results NK92-IL-7R-IM cells exhibited sustained proliferation and high viability independent of exogenous cytokines, superior to IL-2-activated NK cells. This enhanced functionality was driven by the constitutive activation of the JAK/STAT, AKT, and ERK signaling pathways, leading to the upregulation of cytotoxicity-related genes ( GZMA , GZMB ) and anti-apoptotic genes ( BCL2L1 ). In vivo , NK92-IL-7R-IM cells demonstrated significantly potent anti-tumor activity and extended survival compared to control groups. Furthermore, the IL-7R-IM strategy successfully enhanced the function of CAR-NK cells targeting EphA2, EGFR, and CD5 antigens. Conclusions The expression of IL-7R-IM confers cytokine independence and robust anti-tumor activity to NK and CAR-NK cells. This strategy offers a practical solution to improve the persistence and efficacy of off-the-shelf NK cell therapeutics for clinical application.