Carotenoids play important roles in pigmentation, photoprotection, and oxidative stress defense in arthropods. Adult females of the two-spotted spider mite (TSSM), Tetranychus urticae Koch, enter a photoperiodically induced diapause to overwinter. Diapause in TSSM is accompanied by reproductive arrest and the orange body coloration that arises from astaxanthin accumulation. How these two traits are coordinated at the molecular level has long been unclear. Here, we compared the proteomes of adult females reared under diapause-inducing (long-night) and non-diapause-inducing (short-night) photoperiods using LC-MS/MS, followed by RNA interference (RNAi) of candidate genes. A TSSM population collected in Akita, Japan, was maintained under controlled laboratory conditions. Newly hatched larvae were exposed to long-day or short-day photoperiods at 18 °C to induce non-diapause and diapause at the adult stage. The proteome from day 0 to 7 after molting from the teleiochrysalis stage was analyzed using LC-MS/MS. The functional roles of PLAT10 were investigated by RNA interference (RNAi)-mediated gene silencing. RNAi effects were assessed by body and eye pigmentation analysis and HPLC quantification of astaxanthin and its precursor ß-carotene. The carotenoid biosynthesis enzymes phytoene desaturase (TuPDS) and lycopene cyclase/phytoene synthase (TuLCPS), both encoded by genes horizontally transferred from fungi, were more abundant in diapausing females than in non-diapausing females. RNAi of the TuPDS and TuLCPS genes markedly reduced orange pigmentation as well as ß-carotene and astaxanthin contents. This demonstrates that these enzymes are required for diapause- associated pigmentation. Our proteomic analysis further identified a single PLAT (Polycystin-1, Lipoxygenase, Alpha-Toxin) domain protein, TuPLAT10, as one of the most strongly upregulated proteins in diapausing females. The PLAT domain is a lipid-binding module, suggesting a role for TuPLAT10 in lipid metabolism. RNAi of the TuPLAT10 gene restored reproduction under diapause-inducing conditions in addition to the suppression of orange pigmentation. Despite the absence of sequence homology to other carotenoid biosynthesis genes, RNAi of the TuPLAT10 gene selectively reduced TuPDS and TuLCPS protein levels. We propose that TuPLAT10, through its predicted lipid-binding function, couples reproductive arrest and carotenoid pigmentation during diapause in TSSM.