Non-syndromic cleft lip with or without cleft palate (NSCL/P) is a common polygenetic disease. Although genome-wide association studies (GWAS) identified NTN1 gene as a high-priority candidate of NSCL/P, the comprehensive genetic architecture of NTN1 weren’t yet known. Thus, this study aimed to determine full-scale genetic variants of NTN1 for NSCL/P in Chinese Han people. Initially, targeted sequencing of NTN1 gene was performed on 159 NSCL/P patients to identify susceptible single nucleotide polymorphisms (SNPs) associated with NSCL/P. Then, association analysis and burden analysis were separately used to validate the common variants and rare variants identified among large size of samples (1608 NSCL/P cases and 2255 controls). Additionally, NSCL/P subtype association analysis was applied to elucidate the etiology discrepancy of non-syndromic cleft lip with palate (NSCLP) and non-syndromic cleft lip only (NSCLO). Lastly, bioinformatics analysis was performed to annotate and prioritize candidate variants. We found 15 NSCL/P-associated SNPs including rs4791774 (P = 1.10E-08, OR = 1.467, 95% CI: 1.286~1.673) and rs9788972 (P = 1.28E-07, OR = 1.398, 95% CI : 1.235~1.584) originally detected by previous GWASs in Chinese Han ancestry. Four NSCLO risk-associated SNPs and eight specific NSCLP associated SNPs were found. Three SNPs (rs4791331, rs4791774 and rs9900753) were predicted to locate at regulatory region of NTN1. Our study validated the association between NTN1 gene and pathogenesis of NSCL/P and reinforced the hypothesis that NSCLP have a different etiology from NSCLO. We also identified three putative regulatory SNPs in NTN1 gene.