The pine sawyer beetle, Monochamus alternatus Hope, is a devastating wood borer of several species of pine trees, and the main transmitting vector of the pine wood nematode, Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle (Aphelenchida: Parasitaphelenchidae). To explore new techniques for prevention and control of this destructive beetle, a novel gene vacuolar ATPase subunit H (V-ATPase H) was chosen as RNA interference (RNAi) target gene. Relative expression of V-ATPase H in different tissues and silencing efficiency in an in vitro RNAi experiment was assayed by using reverse transcription–quantitative polymerase chain reaction. The results indicated that the mRNA abundance of V-ATPase H in the gut was significantly higher than that in fat body, residual body, and hemolymph. Double-stranded RNA (dsRNA) targeting V-ATPase H was able to silence the expression of target gene effectively at 24 h posttreatment. Expression of immunity-related genes was examined after treatment with dsRNA targeting V-ATPase H, and transcript levels were compared with the control. The results showed that RNAi suppression of V-ATPase H inhibited the expression of immunity-related genes. This is the first demonstration of an in vitro RNAi experiment in any insect hemolymph that provides a novel environment for evaluating RNAi in insects, as well as shows potential for developing RNAi-mediated strategy for the control of M. alternatus.