Polθ-IN-9 is an orally active inhibitor of Polθ polymerase with an IC50 of 9.6 nM and a Kd of 47.5 nM. It demonstrates remarkable selectivity, showing no inhibitory activity against other human DNA polymerases, such as Pol α, Pol ε, Pol γ, Pol λ, and Pol μ. In DLD1 BRCA2 KO cells, Polθ-IN-9 exhibits potent antiproliferative activity with an IC50 of 2.9 μM, and it is highly sensitive to MDA-MB-436 cells with an IC50 of 4.9 μM. In MDA-MB-436 xenograft tumor models, the combination of Polθ-IN-9 with Olaparib enhances DNA damage accumulation, increases γH2AX levels, and inhibits tumor growth. Polθ-IN-9 is useful in studying homologous recombination (HR)-deficient cancers such as breast cancer.