Abstract: To clarify the metabolites and degradation pathways of emamectin benzoate (EB) after trunk injection, the metabolic degradation, metabolites, and their activities of EB in Pinus massoniana were investigated. Residue analysis and metabolite identification were carried out using ultrasonic extraction, silica gel column chromatography, gel column chromatography, preparative chromatography, HPLC, LC-MS, and indoor bioactivity test in P. massoniana after 17 months of trunk application of EB. The retention rate of EB was 50.05%-54.26%. Sixteen metabolites of EB in P. massoniana were isolated and 11 of them were characterized by their molecular formulas: C₄₉H₇₇NO₁₄, C₄₉H₇₅NO₁₄, C₄₁H₆₀O₁₁(MSB_1a), C₄₂H₆₃NO₁₀, C₄₉H₇₇NO₁₄, C₄₉H₇₃NO₁₄(FAB_1a), C₄₈H₇₂O₁₄(4″-S-OH-B_1a), C₄₉H₇₅NO₁₃, C₅₀H₇₇NO₁₃, C₅₀H₇₇NO₁₃ and C₄₉H₇₇NO₁₃. The mortality of Bursaphelenchus xylophilus exposed to the metabolite mixture at 10 mg/L for 24 hours was 69.12%. In the absence of light inside the tree, the metabolism of EB is slower and the catabolic products remain active against pine wood nematodes. This study explains why the dry application of EB can be effective in controlling pine wood nematode disease over a longer period.