The relationship between reproductive diapause and the genes related to vitellogenin (Vg) and its receptor (VgR) in insectoid ovarian development is still unclear. Accordingly, in the present study, we used hematoxylin and eosin staining to study the ovarian structure in the predatory mite Neoseiulus barkeri, a species that shows promise as a biological pest control agent. Staining revealed the presence of oocytes on ovary surfaces, and the oocytes were deposited as yolk granules through the intake of Vg and other nutrients with the development of the ovary. Development of the ovary stopped at the oocyte stage in diapausing adult mites, and this stage presented the same characteristics as the first day of adulthood in non-diapause female adults, where oocytes with nutrient cells, but no yolk granules are observed. In order to further explore the effects of the Vg gene and its receptor on reproduction, the sequences of the N. barkeri vitellogenin genes NbVg1, NbVg2, NbVg3, and NbVgR were analyzed using bioinformatics, and the expression levels of the NbVgs and the VgR at different developmental stages were determined by quantitative polymerase chain reaction (qPCR). The results showed that the NbVgs and NbVgR have complete domains and that the positions of many conservative regions and conservative motif are consistent. The expression levels of the NbVgs and NbVgR were highest in the ovipositional period, followed by those in the preovipositional period. The expression levels of the NbVgs and the VgR in non-diapause female adult mites were significantly higher than those in reproductive diapause female adult mites.