Background: Cross-line treatment with CDK4/6 inhibitors is a challenging topic. Currently, the MAINTAIN study, which involves switching CDK4/6 inhibitors and endocrine therapy, is the only one with positive results. Additionally, in vitro studies have demonstrated that the combination of anti-HER2 agents with CDK4/6 inhibitors, along with fulvestrant, can synergistically enhance inhibition in cell lines with low HER2 expression. The present study aims to assess the efficacy and safety of dalpiciclib, fulvestrant, and pyrotinib in patients with HR-positive and HER2-low advanced breast cancer (BC), who have experienced disease progression after treatment with CDK4/6 inhibitors and AIs. Methods: We propose a single-arm Bayesian optimal phase II design with a time-to-event endpoint. Key eligibility criteria include patients over 18 years of age with HR+, HER2-low advanced BC, disease progression following treatment with CDK4/6 inhibitors and AIs, no more than one line of chemotherapy. Patients who meet these criteria will receive oral dalpiciclib at a dose of 125 mg/day (three weeks on and one week off), intramuscular fulvestrant at a dose of 500 mg every four weeks, and oral pyrotinib at a dose of 320 mg/day until intolerance or disease progression. The primary endpoint of the study is progression-free survival (PFS), and the secondary endpoints include objective response rate (ORR), disease control rate (DCR), overall survival, safety, and biomarker analysis. The study aims to enroll up to 30 patients, and an interim analysis will be conducted after the enrollment of 15 patients to make a go/no-go decision based on the stopping boundaries of the total observation time (TOT). Results: From May 2023 to November 2023, a total of 15 patients were enrolled in the study. The median age of the patients was 50 years (range: 36-72). Among these patients, 7 (46.7%) had visceral metastasis, 11 (73.3%) had undergone previous chemotherapy, 12 (80.0%) received abemaciclib, and 3 (20%) received palbociclib. The median duration of prior treatment with CDK4/6 inhibitors was 14 months (range: 5-33). As of January 24, 2024, a total of 9 patients underwent efficacy evaluation, revealing an ORR of 44.44% (4/9) and a DCR of 66.67% (6/9), 4 patients experienced disease progression. The TOT was 49.50 months, exceeding the predetermined stopping boundary of 22.54 months. Currently, the survival data are immature. The median follow-up period is 3.20 months (95% CI: 2.33-NA), and the median PFS has not been reached. Grade 3 and above treatment-related adverse events were reported in 40% (6/15) of the patients. Conclusions: The preliminary results indicate a certain level of efficacy and manageable safety. Based on these findings, the study will continue to enroll up to 30 participants. Clinical trial information: NCT05806671.