Gamma delta (γδ) T cells are a subset of T lymphocytes expressing γδT cell receptors, which possess both innate and adaptive immune functions. They can recognize and kill tumor cells in a manner independently of MHC restriction. However, their limited quantity in vivo and low efficiency in vitro expansion restrict their clinical application. This study utilized a combination of cytokines interleukin-2 (IL-2), interleukin-15 (IL-15), and the bisphosphonate drug zoledronic acid (Zol) to stimulate γδT cells and optimize their in vitro expansion strategy. Results demonstrated that the combination of Zol (5 μmol/L), IL-2 (100 IU/mL), and IL-15 (10 ng/mL) achieved a 10 000-fold expansion of γδT cells, with a purity of 95.74%among CD3-positive cells. Additionally, metformin treatment significantly increased the proportions of central memory cell subsets from 10.75% to 15.85% and effector memory cell subsets from 5.98% to 19.30% in γδT cells (P<0.01). Metformin also markedly upregulated the expression of anti-tumor factors interferon-γ (IFN-γ), granzyme B (GZMB), and perforin in γδT cells, promoted exosome secretion, and enhanced cytotoxic activity. This study provides a novel strategy for in vitro expansion and functional optimization of γδT cells, laying the foundation for their application in adoptive immunotherapy for tumors.