Previous genome-wide association study of nonsyndromic cleft lip with or without cleft palate (NSCL/P) identified a susceptible variant (rs4791774). We hypothesized that the functional single nucleotide polymorphism (SNP) may be in linkage disequilibrium with this lead SNP. The potential functional SNP (rs4791331) was identified by bioinformatic analysis. A case-control study with 891 orofacial cleft cases and 830 controls was designed to investigate its association with orofacial cleft. The allele-specific DNA-protein binding preference was predicted by JASPAR database. Cell proliferation, cycle and apoptosis, luciferase activity and netrin-1 (NTN1) expression were examined after transfection with the rs4791331 C/T vector in HEK-293 and HEPM cell lines. Forty-six lip tissues of NSCL/P patients were collected to detect NTN1 expression. ntn1a knockout zebrafish models were generated by CRISPR/Cas9 and observed with micro-CT. In the case-control study, the rs4791331-T allele was associated with an increased risk of nonsyndromic orofacial cleft (OR = 1.41, 95% CI = 1.19-1.68), as well as the subgroups cleft lip only (OR = 1.46, 95% CI = 1.14-1.87) and cleft lip and palate (OR = 1.58, 95% CI = 1.27-1.96). The T allele of rs4791331 exhibited anti-apoptotic effects and promoted cell cycle progression at the G1/S transition. Decreased enhancer activity and reduced NTN1 expression following transfection of the T allele were observed. Carriers of the CT/TT genotypes showed significantly lower expression of NTN1 than CC carriers. The ntn1a-/- zebrafish showed relatively wider intermaxillary fissures. These results indicate that rs4791331 (C > T) disrupted motif binding and led to abnormal expression of NTN1, which may be involved in the development of NSCL/P.