Trastuzumab has improved survival rates in human epidermal growth factor receptor 2 (HER2)-positive breast cancer (BC), but drug resistance leads to treatment failure. Natural killer (NK) cell-mediated antibody-dependent cell cytotoxicity (ADCC) represents an essential antitumor immune mechanism of trastuzumab. Traditional Chinese medicine (TCM) has been used for centuries to treat diseases because of its capacity to improve immune responses. Xianling Lianxia formula (XLLXF), based on the principle of “strengthening body and eliminating toxin”, exhibits a synergistic effect in the trastuzumab treatment of patients with HER2-positive BC. Notably, this synergistic effect of XLLXF was executed by enhancing NK cells and ADCC, as demonstrated through in vitro co-culture of NK cells and BC cells and in vivo intervention experiments. Mechanistically, the augmented impact of XLLXF on NK cells is linked to a decrease in cytokine inducible SH2 containing protein (CISH) expression, which in turn activates the Janus kinase 1 (JAK1)/signal transducer and activator of transcription 5 (STAT5) pathway. Collectively, these findings suggested that XLLXF holds promise for enhancing NK cell function and sensitizing patients with HER2-positive BC to trastuzumab.