Recently, the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) updated the guidelines on HER2 testing for invasive breast cancer. Little is known about the impact of the guidelines update. We aimed to study the impact of the 2018 ASCO/CAP HER2 testing guidelines update. We compared the HER2 FISH results interpreted by 2013 and 2018 ASCO/CAP guidelines in 331 cases of invasive breast cancers. We also analyzed the pathological features and clinical outcomes of these cases. In comparing to the 2013 ASCO/CAP guidelines, the HER2 negative rate was increased significantly from 62.5% to 75.8%(P < 0.05), and 13.3% changed from equivocal to negative by the 2018 guidelines. Our findings indicate that the guidelines update significantly increased the rate of negative results. The reclassification of the equivocal results by the 2018 guidelines is the main reason for this change. Patients with HER2 equivocal results were associated with larger tumor size and higher Ki67 index than those with negative results, while clinical outcomes were similar between them.