IRF6 plays an important role in orofacial development. In the present study, we genotyped two polymorphisms (rs642961 and rs2235371) within the IRF6 locus and estimated their associations with risk of nonsyndromic orofacial clefts (NSOC), including the subgroups, in a hospital-based case-control study in a Chinese Han population. In the single locus analyses, we found rs642961 AG and AG/AA genotypes were associated with increased risk of NSOC, especially cleft lip with or without cleft palate (CL/P) and cleft lip with cleft palate (CLP), while significantly decreased risks were associated with rs2235371 CT and CT/TT genotypes. When examining the combined effects of these two polymorphisms and using the rs642961 A and rs2235371 C alleles as the risk alleles, we found genotypes containing 2-4 risk alleles conferred high risk to NSOC, CL/P, and CLP. Furthermore, to test whether rs642961 could modulate IRF6 expression in vivo, we surgically collected lip skin tissues within the adjacent region of lip cleft site and found rs642961 genotypes were associated with differential levels of IRF6 mRNA and protein expression in an allele-dosage manner, providing the first evidence that rs642961 affected IRF6 expression in vivo. Taken together, these findings confirm the contribution of IRF6 genetic variants in the etiology of NSOC in a Chinese Han population.