Background: The presence of tumor-infiltrating lymphocytes (TILs) is associated with better outcomes in human epidermal growth factor receptor 2 (HER2)-positive early breast cancer (EBC) patients who are treated with adjuvant or neoadjuvant trastuzumab-based systemic therapy. Precise subset analysis of TILs is important to understand the role of TILs in the breast tumor microenvironment. We examined the correlation between TILs and treatment outcomes of neoadjuvant dual-HER2 blockage therapy with paclitaxel in HER2-positive EBC. Our aim was to achieve a robust understanding of the predictive value of neuropilin-1 (NRP1), an active CD4 T cell marker, and FOXP3, a well-known regulatory T cell marker, on TILs. Methods: The NeoLath study is a randomized, phase II, five-arm study evaluating the efficacy and safety of lapatinib and trastuzumab followed by lapatinib and trastuzumab plus weekly paclitaxel with or without prolongation of anti-HER2 therapy prior to chemotherapy (18 weeks vs. 6 weeks). The primary endpoint was the comprehensive pathological complete response (pCR) rate. We evaluated the %TILs, %NRP1/TILs and %FOXP3/TILs using core biopsy sections taken at diagnosis, and in surgical specimens after dual HER2 blockage therapy in the prospectively defined, retrospective analysis. Results: A total of 222 tumor samples from 74 patients were used for the present analysis. The mean level of TILs was 20.0% and pCR rate was 56.8%. Univariate analysis showed that %TILs and %NRP1/TILs were associated with pCR and multivariate analysis showed that %NRP1/TILs was significantly associated with pCR (adjusted odds ratio, 1.08 [95% CI, 1.04–1.13]; P < .0001) (table). Conclusions: In addition to TIL evaluation, analysis of NRP1 expression of TILs is important to explore prognosis in HER2-positive EBC patients who are treated with neoadjuvant dual-HER2 blockage therapy for pCR. Clinical trial information: 000007576.