Although artificial insemination has been used for decades in Penaeus monodon aquaculture, the interaction of male and female external genitalia during spermatophore transfer has not been fully documented. As a result, studying the functional anatomy of this process may help to better refine the insemination technique. The sexual act in penaeoid prawns is virtually impossible to observe directly; as a result, this study aimed to describe the functional anatomy and interaction of external genitalia, such as the petasma, appendices masculinae and genital papillae of the male, with that of the thelycum and genital lobes of the female, using a combination of micro-computed tomography and scanning electron microscopy. We hypothesise that the spermatophores are ejaculated into the ventromedial groove of the petasma and squeezed by abdominal flexure into the spermathecae of female's thelycum. During this process, the 'spike-like' setae observed on the petasma and appendices masculinae are speculated to control the transferring direction of the spermatophores. The approach of three dimensional remodelling and animation reported in this study may prove useful in the examination of further hypotheses related to the functional anatomy of external genitalia and/or appendages for other crustacean species.