Background: High levels of circulating tumor cells (CTCs) at initiation of therapy in patients with metastatic breast cancer (MBC) are associated with worse prognosis. In patients with hormone receptor–positive (HR+), HER2-negative MBC, the benefit of maintaining CDK4/6 inhibitor (CDK4/6i) after progression on first-line therapy with endocrine therapy and CDK4/6i is not fully defined. To study the relationship between CTCs, tumor characteristics, and clinical outcomes, we enumerated CTC dynamics in patients with HR+ HER2- MBC enrolled on a phase II, multi-center, open-label interventional trial of palbociclib and fulvestrant after disease progression on prior palbociclib with an aromatase inhibitor (NCT02738866). Methods: Blood samples from study participants were collected at baseline (pretreatment), cycle 2 (C2, 8 weeks after initiation of fulvestrant and palbociclib), and at disease progression. Using a previously analytically validated assay (CellSearch), CTCs were enumerated. Progression-free survival (PFS) was estimated using the Kaplan Meier method. The relationship between CTC count at baseline (< 5, >5/7.5 ml Whole Blood (WB)) and PFS was analyzed using Cox proportional hazards regression. The association between PFS and CTC count among patients with baseline CTC ≥5 was estimated with a Cox proportional hazards model, adjusting for age, race, and progesterone receptor (PgR). Results: Between May 2017 and September 2022, baseline samples were collected from 52 of 60 trial participants enrolled at 5 centers in the United States, mean age 55 years (SD 14). Baseline CTC counts were <5 in 30/52 (58%) and >5 in 22/52 (42%) of participants. Among participants at baseline, only tissue PgR negativity was associated with higher likelihood of having elevated CTCs >5 (p = 0.04). Association of CTCs >5 and select patient and tumor characteristics is shown in Table 1. There was no statistically significant association between continuously measured CTC and PFS among those with elevated CTCs at baseline. Median duration of therapy among the entire 52 participant cohort was 4 months. Median PFS in participants with baseline CTC <5 vs >5/7.5 ml WB was 7.83 (95% CI [5.37, 12.60]) and 2.83 (95% CI [1.77, 4.73]) months, respectively (HR): 2.10, 95% CI [1.15, 3.82], p = 0.01). Conclusions: Baseline CTCs >5/7.5 ml WB conferred a 2-fold increased risk of disease progression. Tissue PgR negativity was associated with baseline CTCs >5. These data can be used to inform new studies in which baseline and early change in CTCs may inform second line treatment decisions in women with HR+, HER2- MBC.