Mentha canadensisL. is an important spice crop and medicinal herb with high economic value. The plant is covered with peltate glandular trichomes, which are responsible for the biosynthesis and secretion of volatile oils. Plant non-specific lipid transfer proteins (nsLTPs) belong to a complex multigenic family involved in various plant physiological processes. Here, we cloned and identified a non-specific lipid transfer protein gene (McLTPII.9) fromM. canadensis, which may positively regulate peltate glandular trichome density and monoterpene metabolism.McLTPII.9was expressed in mostM. canadensistissues. The GUS signal driven by theMcLTPII.9promoter in transgenicNicotiana tabacumwas observed in stems, leaves, and roots; it was also expressed in trichomes.McLTPII.9was associated with the plasma membrane. Overexpression ofMcLTPII.9in peppermint (Mentha piperita.L) significantly increased the peltate glandular trichome density and total volatile compound content compared with wild-type peppermint; it also altered the volatile oil composition. InMcLTPII.9-overexpressing (OE) peppermint, the expression levels of several monoterpenoid synthase genes and glandular trichome development-related transcription factors—such as limonene synthase (LS), limonene-3-hydroxylase (L3OH), geranyl diphosphate synthase (GPPS),HD-ZIP3, andMIXTA—exhibited varying degrees of alteration.McLTPII.9overexpression resulted in both a change in expression of genes for terpenoid biosynthetic pathways which corresponded with an altered terpenoid profile in OE plants. In addition, peltate glandular trichome density was altered in the OE plants as well as the expression of genes for transcription factors that were shown to be involved in trichome development in plants.