Nonsyndromic oral cleft is a common developmental malformation of humans. Embryonic development is regulated by microRNAs. MicroRNA-140-5p (miR-140-5p) was found to regulate palatal development. As sequence variants in miRNA genes are likely to affect miRNA expression and/or maturation, we investigated the miRNA-140 gene and identified a SNP (rs7205289: C>A) located in precursor miRNA-140. We carried out a case–control analysis in 557 patients with nonsyndromic oral clefts and 306 unaffected controls from west China and found that the frequency of minor allele (A allele) was significantly increased (P = 0.003 after Bonferroni correction) in nonsyndromic cleft palate (NSCP) patients in comparison with that in controls. We constructed expression vectors of primary miRNA-140 (pri-miR-140) with the major and minor alleles of rs7205289. The vectors were transfected into HEK293 cells, and the mature forms of miR-140 were detected by Northern blot. Compared to the vector with the C allele, the vector with the A allele was found to influence the miR-140 processing, resulting in a significant decrease of miR-140-5p and an increase of miR-140-3p. These results suggest that the SNP located in pre-miR-140 contributes to NSCP susceptibility by influencing the processing of miR-140. © 2010 Wiley-Liss, Inc.