Abstract Strumigenys hexamera (Brown) is a terrestrial ant nesting exclusively in the soil and specialized in predation on diplurans and small-sized myriapods (symphyllans and centipedes). Its colony is monogynous and previous dissections of the queens had suggested that they were uninseminated, thus they could reproduce thelytokously. In the current study, first, the spermathecae of the queens (dealate gynes) from worker-containing colonies were examined histologically in details. All specimens examined had no spermatozoa in the spermatheca. Second, alate gynes were reared with workers in the laboratory and whether they lay eggs that produce female offspring was studied. In all of 10 culture replicates, new workers were produced from eggs those gynes had laid. No spermatozoa in their spermathecae and no ovary in the worker caste were confirmed histologically after rearing experiments. These results reveal that S. hexamera is thelytokous at least for a studied population in central Japan.