Trichome initiation and patterning are controlled by the TTG1–bHLH–MYB regulatory complex. Several MYB transcription factors have been determined to function in trichome development via incorporation into this complex. This study examined the role of MYB82, an R2R3-MYB transcription factor, in Arabidopsis trichome development. MYB82 was revealed to be a nuclear-localized transcription activator. Suppression of MYB82 function by fusion with a dominant repression domain (SRDX) resulted in glabrous leaves, as did overexpression of N-terminal-truncated MYB82. Overexpression of MYB82 genomic sequence, but not its cDNA sequence, led to reduced trichome numbers. Further investigation indicated that at least one of the two introns in MYB82 is essential to the protein’s trichome developmental function. An MYB-binding box was identified in the third exon of MYB82, which was inferred to be crucial for MYB82 function because the mutation of this box interfered with the ability of MYB82 to rescue the gl1 mutant. Protein interaction analysis revealed that MYB82 physically interacts with GLABRA3 (GL3). In addition, MYB82 and GL1 can form homodimers and heterodimers at R2R3-MYB domains, which may explain why their overexpression reduces trichome numbers. These results demonstrate the functional diversification of MYB82 and GL1 in trichome development.