Trichomes are universally present in plants and trichome development is delicately regulated. Trichomes are responsible for pubescence, and their density is associated with agronomical traits related to insect resistance, evapotranspiration and yield. Almost a century ago, three dominant alleles related to pubescence density in soybean, namely Pd1 (dense pubescence), Ps (sparse pubescence) and P1 (glabrous), were identified. However, their identity and inheritance remain unclear. Here, through genome-wide association study (GWAS) and map-based cloning, we determined the genetic basis of these three traits. The sparse pubescence phenotype of Ps was attributed to a copy number variation (CNV) of a 25.6-kb sequence that includes a gene encoding a protein with WD40 and RING domains, the dense pubescence phenotype of Pd1 was attributed to a T-C transition in the last exon of a HD-Zip transcription factor gene, and the glabrous phenotype of P1 was attributed to a G-A transition in the first exon of a lipid transfer protein gene. Genetic and biochemical analyses revealed that Pd1 functions as a transcriptional activator that binds to the promoters of the P1 and Ps genes to induce their expression and that binds to its own promoter to act as a transcriptional inhibitor. In addition, Ps can interact with Pd1 and weaken the transcriptional activity of Pd1. Taken together, our results demonstrate that Pd1, Ps, and P1 form a complex feedback loop to regulate pubescence formation.