The effects of long-term nicotine exposure on the treatment of HER2-positive breast cancer have only been studied epidemiologically, leaving the molecular mechanisms unclear. Therefore, it's challenging for clinicians to devise effective treatments based on this correlation. Our investigation explores the effects of prolonged nicotine exposure on the interaction between α9-nAChR and HER2 in HER2-positive breast cancer cells, highlighting its impact on anti-HER2 drug therapy efficacy. We used fluorescence resonance energy transfer (FRET) analysis to compare HER2 + tumor samples from smokers and non-smokers, finding a more vital interaction between α9-nAChR and HER2 in non-smoker's breast tumor tissues. A simulated study on the breast cancer patient-derived xenograft (PDX) tumor mouse model (n = 6 per group) revealed that long-term nicotine exposure significantly reduced this interaction compared to controls. Due to the poorer response of low HER2 tumors to trastuzumab in clinical practice, most patients require combination chemotherapy and have a worse prognosis. Therefore, this study chose HER2-low PDX models to simulate nicotine exposure and explore treatment strategies. Mice subjected to prolonged nicotine exposure exhibited faster tumor growth and reduced trastuzumab efficacy compared to controls (*p <
0.05). Our results demonstrate that extended nicotine exposure weakens α9-nAChR and HER2 interaction, reducing the effectiveness of HER2-targeted therapy, fostering drug resistance, and underscoring the carcinogenic risks of nicotine.