Diapause is a physiological adaptation that allows an organism to survive adverse environmental conditions. Diapause occurs at a specific developmental stage in each species. There are few reports regarding the molecular regulatory mechanism of nymphal diapause in Laodelphax striatellus, which is an important graminaceous crop pest. Our previous studies identified the conditions for nymphal diapause in this species. Here, we combined RNA sequencing transcriptomics and quantitative proteomic analyses to identify nymphal diapause-related genes and proteins. The analysis of differentially regulated genes identified four gene/protein pairs that were synchronously up-regulated, and six gene/protein pairs that were synchronously down-regulated, suggesting that these genes may regulate nymphal diapause. The up-regulated gene juvenile hormone acid methyl transferase (JHAMT) and the down-regulated gene cytochrome P450 monooxygenase (CYP314A1, Shd) were chosen for further functional studies. After knocking-down of LsJHAMT and LsShd in vivo by RNA interference, the titer of JH III and 20E decreased significantly, and the duration of the nymphal development period was severely altered. Thus LsJHAMT and LsShd regulated JH III and 20E titers in the hemolymph to control the nymphal diapause status. This study may lead to new information on the regulation nymphal diapause of this important agricultural insect pest.