Ras opposite (Rop) is known to play an essential role in regulating vesicle trafficking, including synaptic transmission and general secretion. The fundamental roles of Rop have been confirmed by the observation that null mutations in many organisms generate lethal phenotypes during embryogenesis. However, the effects of Rop during the postembryonic stages, especially in non‐model organisms, remain largely unknown. Here, we provide new data that enhance our understanding of Rop's roles in the adults of multiple species of Tetranychus spider mites (Acari: Tetranychidae), a class of notorious agricultural pests. Our in silico and experimental evidence demonstrated that Rop is under purifying selection and is highly conserved in Tetranychus spp. RNA interference experiments showed that Rop is required for maintaining normal fecundity but has no significant effect on survival. We further demonstrate that knockdown of Rop darkens the body color of spider mites and blocks the excretion of fecal pellets, which is likely to be related to an abnormality in the excretion of food waste in the digestive system. Overall, our findings clarify novel functions of a vesicle trafficking‐related gene in the adult stage of multiple Tetranychus species and highlight the need to evaluate the roles of essential genes in various organisms. Rop is under strong purifying selection and is highly conserved in spider mites. Rop RNA interference decreases female fecundity, darkens the body color, and blocks the excretion of fecal pellets.