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Trichomes are specialized epidermal cells that play multiple roles in plant development. However, knowledge about the molecular mechanism of trichome development in rice is limited. In this study, a hairy leaf locus HL6 SWWR of rice variety Suwangwanger (SWWR) was identified by map-based cloning. Transgenic complementation and knock-out mutation demonstrated that HL6SWWR regulated trichomes on leaves and expression levels of HL6SWWR affected trichome elongation. Transgenic experiments of PROSWWR:HL6NIP and PRONIP:HL6SWWR indicated that the promoter and coding sequences of HL6SWWR were indispensable for trichome regulation. Sequencing alignment of the promoter and coding regions of HL6 in 22 varieties showed that some cis-binding elements in the promoter region may be related to trichome development, while no decisive variation was found in the coding sequence. Furthermore, RNA-sequencing analysis revealed that 1 415 differential expression genes (DEGs) were detected in hl6SWWR mutant vs wild type and that 1 010 DEGs were found in HL6SWWR complementary transgenic line vs wild type. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that most of the DEGs were involved in metabolite pathway, secondary metabolite biosynthesis, plant pathogen interaction and phytohormone signal transduction in both the two groups. The results indicated that these enhanced pathways are critical during trichome development in rice. Taken together, our results provided new views into the regulatory mechanism of trichome formation in rice.