AbstractGermline PALB2 pathogenic variants are associated with an increased lifetime risk for breast, pancreatic, and ovarian cancer. However, the interpretation of the pathogenicity of numerous PALB2 missense variants of uncertain significance (VUSs) identified in germline genetic testing remains a challenge. Here we selected ten potentially pathogenic PALB2 VUSs identified in 2279 Chinese patients with breast cancer and evaluated their impacts on PALB2 function by systematic functional assays. We showed that three PALB2 VUSs p.K16M [c.47 A > T], p.L24F [c.72 G > C], and p.L35F [c.103 C > T] in the coiled-coil domain impaired PALB2-mediated homologous recombination. The p.L24F and p.L35F variants partially disrupted BRCA1-PALB2 interactions, reduced RAD51 foci formation in response to DNA damage, abrogated ionizing radiation-induced G2/M checkpoint maintenance, and conferred increased sensitivity to olaparib and cisplatin. The p.K16M variant presented mild effects on BRCA1-PALB2 interactions and RAD51 foci formation. Altogether, we identify two novel PALB2 VUSs, p.L24F and p.L35F, that compromise PALB2 function and may increase cancer risk. These two variants display marked olaparib and cisplatin sensitivity and may help predict response to targeted therapy in the clinical treatment of patients with these variants.