There exist gender differences in the modulation of catechol-O-methyltransferase (COMT) Val158Met polymorphism on cognitive performance; however, the underlying gene-anatomy-cognition pathways remain unknown. Here we hypothesize that prefrontal volume may mediate the modulation of COMT Val158Met polymorphism on interference resolution capacity in a gender-dependent manner. In 261 healthy young human subjects (143 males and 118 females), a 2-way analysis of variance showed a COMT × gender interaction (P = 0.023) on interference resolution capacity. Val/Val subjects performed worse in Stroop interference test than Met/Met subjects only in males (P = 0.028). Voxel-wise analysis in the whole brain also exhibited a COMT × gender interaction on gray matter volume (GMV) in the left lateral frontal pole (FP). Val/Val male individuals exhibited significantly decreased GMV in the left lateral FP than Val/Met (P = 0.003) and Met/Met (P = 0.006) male carriers. Mediation analysis revealed that the GMV of the left lateral FP mediated the association between COMT polymorphism and interference resolution in males. These findings provide a gene-anatomy-cognition pathway to describe how COMT Val158Met polymorphism affects interference resolution capacity via modulating the prefrontal GMV in healthy male subjects.