BACKGROUND: The unmet needs of managing patients with hormone receptor-positive/human epidermal growth factor receptor 2-negative (HR+/HER2-) breast cancer who progress after cyclin-dependent kinase (CDK)4/6 inhibitor (CDK4/6i) treatment remain unclarified. METHODS: This was a phase 1b/2, single-arm, open-label study that enrolled 29 patients with HR+/HER2- breast cancer who experienced first-line palbociclib treatment failure. The primary endpoint was the incidence of dose-limiting toxicity (DLT). The secondary endpoints were the objective response rate (ORR) and progression-free survival (PFS). The clinical trial registration number is ClinicalTrials.gov: NCT05953350. FINDINGS: The phase 1b study demonstrated no DLT in patients treated with hydroxychloroquine (HCQ; 600 mg, bis in die [bid]) plus increasing doses of palbociclib (100, 150, or 200 mg, quaque die [qd]). The plasma pharmacokinetics of palbociclib were not significantly affected by HCQ. The recommended phase 2 dose (RP2D) was HCQ (600 mg, bid) plus palbociclib (200 mg, qd). The dose-expansion cohort demonstrated that HCQ plus palbociclib (200 mg, qd) treatment was tolerable. Grade 3 treatment-emergent adverse events (TEAEs) with an incidence higher than 15.0% included neutropenia (25.0%), leukopenia (25.0%), fatigue (20.0%), and back pain (15.0%). The ORR of all enrolled patients in our present trial was 41.4% (12/29). In the dose-expansion cohort, with the last enrolled patient having a follow-up duration of 32.3 weeks, the median PFS was not reached. The clinical benefit rate (CBR) at 6 months was 90.0% (95% confidence interval [CI]: 68.3%-98.8%). These findings were supported by preclinical data. CONCLUSIONS: Combined HCQ with high-dose CDK4/6i palbociclib (200 mg, qd) showed tolerable toxicity and promising efficacy for patients with advanced HR+/HER2- breast cancer after CDK4/6i failure. FUNDING: This work was funded by the National Natural Science Foundation of China.