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Cytochrome b5 (Cyt-b5) is a small heme protein and known to be involved in a wide range of biochemical transformations, including cytochrome P450 monooxygenase (CYP)-mediated metabolism of endogenous and exogenous compounds. Studies on Cyt-b5 are more concentrated in mammals, but are relatively rare in insects. The characteristics and function of Cyt-b5 from Locusta migratoria have not been described yet. We sequenced the full-length cDNA sequence of Cyt-b5 from L. migratoria (LmCyt-b5) by reverse transcription-PCR (RT-PCR) based on locust transcriptome database. The phylogenetic analysis showed that LmCyt-b5 was closely related to the Cyt-b5 from Blattodea. LmCyt-b5 was highly expressed in ovary, Malpighian tubules, midgut, gastric caeca, and fat bodies. Silencing of LmCyt-b5 had no effect on the susceptibility of L. migratoria to four different insecticides. Suppression of LmCyt-b5 or silencing of both LmCyt-b5 and LmCPR did not significantly change the total CYP activity toward the substrate 7-ethoxycoumarin (7-EC). However, coexpression of LmCYP6FD1 with LmCPR and LmCyt-b5 together in Sf9 cells by using Bac-to-Bac baculovirus expression system significantly increased the catalytic activity of LmCYP6FD1 toward 7-EC as compared with the coexpression of LmCYP6FD1 with cytochrome P450 reductase (LmCPR) or LmCyt-b5 separately. These results suggest that LmCyt-b5 plays an important role in the catalytic reaction of LmCYP6FD1 toward 7-EC in our in vitro experiments. Further study is needed to clarify the role of LmCyt-b5 in CYP-mediated catalytic reactions in L. migratoria.