Maize unilateral cross-incompatibility (UCI) is a pre-zygotic reproductive barrier known to restrict gene flow between maize and teosinte and among maize populations, which has practical application for isolation of maize varieties of different kinds in commercial breeding programs. Gametophyte factor1 (Ga1), Gametophyte factor2 (Ga2), and Teosinte crossing barrier1 (Tcb1) are three distinct UCI loci. Though great efforts have been made to identify their causative genes, the Tcb1 male determinant still remains unidentified. Here, we demonstrate that a pectin methylesterase (PME) gene, Tcb1-m, functions as male determinant at the Tcb1 locus. Expressing Tcb1-m endows tcb1 pollen with the ability to penetrate the female barrier of Tcb1-S. Tcb1 and Ga1 are genetically linked in teosinte, and diversification of them appears to have occurred after maize domestication. We prove the feasibility of stacking different UCI loci can strengthen their applications in maize production. The lack of interaction between the male and female determinants of maize UCI indicates a distinct mechanism from that of self-incompatibility (SI). Our study lays a solid foundation for deciphering the identity and specificity of maize UCI.