Japanese pine sawyer, Monochamus alternatus, is the main pest that mediates pine wilt nematode, Bursaphelenchus xylophilus, that causes serious damage to pine forests. In this study, we studied the strategy to control M. alternatus using entomopathogenic fungi. The fungi were collected from soil by an insect-baiting method and two fungal isolates (Metarhizium anisopliae JEF-197 and JEF-279) showed high virulence against M. alternatus. The Metarhizium isolates were evaluated for insecticidal activity against M. alternatus by spray treatment on live pine trees and wintering trees, and the M. anisopliae JEF-197 showed high insecticidal activity. In addition, the interaction of fungi and M. alternatus were analyzed by RNA-seq. This result can contribute to the development of insect control agents using entomopathogenic fungi.