Orofacial clefts (OFCs) are the most common congenital craniofacial disorders and cause serious problems with the appearance, orofacial function and mental health of the patients. The fibroblast growth factor (FGF) signaling pathway is critical for several aspects of craniofacial development and loss-of-function mutations of coding genes for multiple FGFs and FGFRs can lead to OFCs. We recently characterized FAM3B as a novel ligand of FGF signaling, which, through binding to FGFRs and activating downstream ERK, regulates craniofacial development in Xenopus. In this study, we identify two rare variants in FAM3B (p.Q61R and p.D128G) via target region sequencing of FAM3B on 144 unrelated sporadic patients with non-syndromic OFCs (NSOFCs). Bioinformatic analysis predict that these two variants are likely to be damaging and biochemical experiments show that these two variants weaken the FGF ligand activity of FAM3B by decreasing its expression and thus secretion. In summary, our results indicate that FAM3B is a novel candidate gene for NSOFCs in humans.