Key message Map-based cloning revealed that two novel soybean distorted trichome mutants were due to loss function of GmNAP1 gene, which affected the trichome morphology and pavement cell ploidy by regulating actin filament assembly. Abstract Trichomes increase both biotic and abiotic stress resistance in soybean. In this study, Gmdtm1-1 and Gmdtm1-2 mutants with shorter trichomes and bigger epidermal pavement cells were isolated from an ethyl methylsulfonate mutagenized population. Both of them had reduced plant height and smaller seeds. Map-based cloning and bulked segregant analysis identified that a G-A transition at the 3ʹ boundary of the sixth intron of Glyma.20G019300 in the Gmdtm1-1 mutant and another G-A transition mutation at the 5ʹ boundary of the fourteenth intron of Glyma.20G019300 in Gmdtm1-2 ; these mutations disrupted spliceosome recognition sites creating truncated proteins. Glyma.20G019300 encodes a Glycine max NCK-associated protein 1 homolog ( GmNAP1 ) in soybean. Further analysis revealed that the GmNAP1 involved in actin filament assembling and genetic information processing pathways during trichome and pavement cell development. This study shows that GmNAP1 plays an important role in soybean growth and development and agronomic traits. Electronic supplementary material The online version of this article (10.1007/s11103-020-01013-y) contains supplementary material, which is available to authorized users.