Single-nucleotide polymorphisms (SNPs) in protein-coding regions of genes which were previously reported to be associated with nonsyndromic cleft lip, with or without palate involvement (NSCL/P), were investigated. Twelve candidate loci [platelet-derived growth factor C (PDGFC), platelet-derived growth factor subunit A (PDGFA), platelet-derived growth factor receptor alpha (PDGFRA), glycine receptor alpha 2 (GLRA2), glycine receptor beta (GLRB), ATP binding cassette subfamily A member 4 (ABCA4), MAF bZIP transcription factor B (MAFB), interferon regulatory factor 6 (IRF6), CCDC26 long non-coding RNA (CCDC26), paired box 7 (PAX7), ventral anterior homeobox 1 (VAX1), and netrin 1 (NTN1)] covering 1.5 Mbp were sequenced in 136 NSCL/P patients and 54 healthy controls. Twenty-five genomic variants identified were further validated in another 400 NSCL/P and 200 controls. Two SNPs in IRF6 showed a protective effect against the development of NSCL/P (rs12405750, OR = 0.54, 95% CI: 0.41-0.69; and rs2235371, OR = 0.55, 95% CI: 0.43-0.71). The missense variant, rs2235371, alters the conserved amino acid valine to isoleucine at codon 274 (V274I). We observed that SNPs at IRF6 (rs2235371 and rs12405750) and GLRB (rs73856838 and rs72685584) show consistent interaction effects. The association between the missense SNP rs2235371 in gene IRF6 and NSCL/P suggests that this SNP may play an important role as a risk factor for NSCL/P in the Han Chinese populations. The marginal signal near 4q31 detected in previous genome-wide association studies might be caused by an interaction between the IRF6 and GLRB genes. This interaction needs to be further validated by experimentation in follow-up studies.