Background: Our previous MUKDEN 01 study showed a promising total pathological complete response (tpCR) rate of 30.4% with neoadjuvant pyrotinib plus dalpiciclib (cyclin dependent kinase 4/6 inhibitor) and letrozole in patients with triple-positive breast cancer (TPBC), but the efficacy still needs improvement. This pilot study explored the efficacy and safety of adding trastuzumab to this neoadjuvant regimen in TPBC. Methods: Patients received five 28-day cycles of pyrotinib (320 mg once daily), dalpiciclib (125 mg once daily on days 1-21) and letrozole (2.5 mg once daily), plus six 21-day cycles of trastuzumab (8 mg/kg loading dose followed by 6 mg/kg maintenance dose on day 1), followed by surgery. The primary endpoint was tpCR (ypT0/is, ypN0) rate. Secondary endpoints were objective response rate (ORR), breast pathological complete response (bpCR; ypT0/is) rate, residual cancer burden (RCB), change in Ki-67 level from baseline to surgery, and safety. Results: Between February 16, 2022 and June 2, 2022, 12 patients were enrolled. Seven (58%; 95% CI, 27.7-84.8) patients achieved tpCR and bpCR. The rate of RCB 0-I was 75% (95% CI, 46.8-91.1%). ORR was 92% (95% CI, 64.6-98.5%). Mean Ki-67 level was significantly reduced from 45.0% (95% CI, 19.5% - 70.5%) at baseline to 17.2% (95% CI, 0.7% - 33.7%) after neoadjuvant therapy (P<0.05). The most common grade 3 adverse events were diarrhea (4 [33%]) and decreased neutrophil count (3 [25%]). No grade 4 events or treatment-related deaths occurred. Conclusions: This four-drug neoadjuvant regimen shows promising pathological response in patients with TPBC, with an acceptable safety profile. The results warranted further validation. Clinical trial information: NCT05228951.