BackgroundThe flesh fly,Sarcophaga peregrina(Diptera: Sarcophagidae), is an important hygiene pest, that causes myiasis in humans and other mammals, typically livestock, and as a vector for various parasitic agents, including bacteria, viruses, and parasites. The role of long non-coding RNAs (lncRNAs) in regulating gene expression during metamorphosis of the flesh fly has not been well established.Methodology/Principal findingsIn this study, we performed genome-wide identification and characterization of lncRNAs from the early pupal stage (1-days pupae), mid-term pupal stage (5-days pupae), and late pupal stage (9-days pupae) ofS.peregrinaby RNA-seq, and a total of 6921 lncRNAs transcripts were identified. RT-qPCR and enrichment analyses revealed the differentially expressed lncRNAs (DE lncRNAs) that might be associated with insect metamorphosis development. Furthermore, functional analysis revealed that the DE lncRNA (SP_lnc5000) could potentially be involved in regulating the metamorphosis ofS.peregrina. RNA interference ofSP_lnc5000caused reduced expression of metamorphosis-related genes in 20-hydroxyecdysone (20E) signaling (Br-c, Ftz-F1), cuticle tanning pathway (TH, DOPA), and chitin related pathway (Cht5). Injection of dsSP_lnc5000in 3rd instar larvae ofS.peregrinaresulted in deformed pupae, stagnation of pupal-adult metamorphosis, and a decrease in development time of pupal, pupariation rates and eclosion rates. Hematoxylin-eosin staining (H&E), scanning electron microscope (SEM) observation and cuticle hydrocarbons (CHCs) analysis indicated thatSP_lnc5000had crucial roles in the metamorphosis developmental by modulating pupal cuticular development.Conclusions/SignificanceWe established that the lncRNASP_lnc5000potentially regulates the metamorphosis ofS.peregrinaby putatively affecting the structure and composition of the pupal cuticle. This study enhances our understanding of lncRNAs as regulators of metamorphosis inS.peregrina, and provide valuable insights into the identification of potential targets for vector control and the development of effective strategies for controlling the spread of myiasis and parasitic diseases.