Abstract: 【Aim】This study aims to enrich the information on the physical and chemical properties, and molecular characteristics of peroxiredoxin-5 (AmPRX5) from the western honeybee, Apis mellifera, investigate the expression pattern of AmPRX5, and explore the role of AmPRX5 in antioxidant stress response in Apis mellifera following infection with Ascosphaera apis, so as to provide a scientific basis for further research on the function and action mechanism of AmPRX5. 【Methods】The CDS of AmPRX5 of Apis mellifera was amplified by PCR. Relevant software was used to predict the physicochemical properties and molecular characteristics of AmPRX5, construct the protein interaction network, identify conserved motifs and domains, and conduct phylogenetic analysis. RT-qPCR was employed to detect the expression levels of AmPRX5 in different developmental stages (egg, 3- and 5-day-old larvae, 1- and 2-day-old prepupa, 1-day-old pupa, and 1-, 2-, 6-, 8- and 12-day-old adults) of workers and different tissues (brain, hypopharyngeal gland, midgut, antenna, venom gland, cuticle and fat body) of worker adults and the guts of the 4-, 5- and 6-day-old larvae of Apis mellifera after inoculation of the 3-day-old larvae with Ascosphaera apis. The hydrogen peroxide (H₂O₂) contents in the guts of the 4-, 5- and 6-day-old larvae after inoculation of the 3-day-old larvae with Ascosphaera apis were detected by spectrophotometry. 【Results】The CDS of AmPRX5 of Apis mellifera was successfully cloned, with the length of 558 bp. AmPRX5 has the molecular formula of C₉₀₄H₁₄₄₅N₂₄₁O₂₆₄S₈, the molecular weight of approximately 20.17 kD, an isoelectric point of 8.99, and the average hydrophilic coefficient of -0.013. AmPRX5 contains 19 phosphorylation sites, and does not contain typical signal peptides and transmembrane domains. The secondary structure of AmPRX5 comprises 88 random coils, 59 α-helices and 38 extended chains. AmPRX5 forms an interaction network with 10 proteins, including thioredoxin reductase. PRX5 proteins from Apis mellifera and nine other bee species all contain an identical PRX5 domain and six conserved motifs. The PRX5 from Apis mellifera shares the highest homology with that from Apis cerana. The expression level of AmPRX5 in the 1-day-old pupae was the highest and significantly higher than those in the egg, 3-day-old larva, 5-day-old larva, 1-day-old prepupa and 2-day-old prepupa. The expression level of AmPRX5 in the 12-day-old adults was the highest and significantly higher than those in the 1-, 2-, 6- and 8-day-old adults. The expression level of AmPRX5 in the adult antenna was the highest and significantly higher than those in the midgut, fat body, cuticle, venom gland and brain. After inoculation of the 3-day-old larvae of Apis mellifera with Ascosphaera apis, the expression level of AmPRX5 in the gut of the 4-day-old larvae was up-regulated but showed no significant difference from that in the uninoculated control group, that in the gut of the 5-day-old larvae was extremely significantly up-regulated and that in the gut of the 6-day-old larvae was significantly up-regulated as compared with those in the uninoculated control group. After inoculation of the 3-day-old larvae with Ascosphaera apis, the H₂O₂ contents in the guts of the 4- and 5-day-old larvae increased extremely significantly and that in the gut of the 6-day-old larvae increased significantly as compared with those in the uninoculated control group. 【Conclusion】AmPRX5 is a potential hydrophilic, non-transmembrane and intracellular protein. AmPRX5 is expressed dynamically and differentially in different developmental stages and tissues of Apis mellifera. AmPRX5 may participate in the response of larvae to the infection of Ascosphaera apis by influencing the H₂O₂ content.