Background Head louse females secrete liquid gel, which is mainly composed of the louse nit sheath protein 1 (LNSP1) and LNSP2, when they lay eggs. The gel is crosslinked by transglutaminase (TG) to form the nit sheath, which covers most part of egg except the top operculum area where breathing holes are located. Knowledge on the selective mechanism of nit sheath solidification to avoid uncontrolled crosslinking could lead to design a novel way of louse control, but no information is available yet. Methods To elucidate the crosslinking mechanisms of nit sheath gel inside the reproductive system of head louse females, in situ hybridization in conjunction with microscopic observation of the oviposition process was conducted. Results Histochemical analysis revealed that LNSP1 and LNSP2 are expressed over the entire area of accessory gland and uterus, whereas TG expression site is confined to a highly localized area around the opening of posterior oviduct. Detailed microscopic observations of oviposition process uncovered that a mature egg is positioned in the uterus after ovulation. Once aligned inside the uterus, the mature egg is redirected so that its operculum side tightly held by the ventral end of uterus being positioned toward the head again and its pointed bottom end being positioned toward the dorsal end of uterus, which functions as a reservoir for the nit sheath gel. Conclusions Physical separation of the TG-mediated crosslinking site from the ventral end of uterus is necessary to avoid uncontrolled crosslinking inside the uterus and to ensure selective crosslinking over only the lower part of egg without any unwanted crosslinking over the operculum during oviposition.