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.