Background Non-syndromic isolated cleft lip with or without cleft palate (NCL/P) is a common congenital anomaly in humans, the aetiology of which is complex and associated with both genetic and environmental factors. It has been reported that maternal nutritional factors are likely to play a major role in development of NCL/P in the embryo. Objective As the mechanism by which folic acid and choline supplementation prevents NCL/P is poorly understood, the relationship between 16 polymorphic variants of 12 genes encoding enzymes involved in the metabolism of these two nutrients and the risk of facial clefts was investigated. Results It was found that individuals with the AA genotype of the BHMT rs3733890 polymorphism have a significantly lower risk of orofacial clefts (OR 0.1450, 95% CI 0.0420 to 0.4995; p=0.0005; pcorr=0.008). It was also demonstrated that the rs7639752 polymorphism of the PCYT1A gene increases the risk of NCL/P nearly twofold in the Polish population (OR 1.891, 95% CI 1.151 to 3.107; p=0.011), but this association would not withstand correction for multiple testing (pcorr=0.176). The genetic variations in CBS, MTHFD1, MTHFR, MTR, MTRR, TCN2, BHMT2, CHDH, CHKA, and PEMT were not separately correlated with NCL/P risk. However, the Multifactor Dimensionality Reduction (MDR) analysis showed a significant epistatic interaction between MTHFR (rs1801133), MTR (rs1805087), and PEMT (rs4646406) in NCL/P susceptibility. Conclusion This study demonstrates that choline metabolism may play an important role in the aetiology of NCL/P. Polymorphic variants of BHMT and PCYT1A and interactions between genes of choline and folate metabolism might influence the risk of NCL/P in the Polish population.