[Aim]  To provide a reference and foundation for functional research on the Apis mellifera caspase-3 gene (Am-caspase-3). [Methods]  The CDS of Am-caspase-3 was amplified with RT-PCR and verified using Sanger sequencing. The physicochemical properties, protein structure and protein interaction network of the Am-caspase-3 protein were then analyzed with bioinformatics software. A phylogenetic analysis of caspase-3 was conducted by using MEGA software. RT-qPCR was used to detect the expression of Am-caspase-3 in different tissues of adult workers infected with Nosema ceranae, and in worker larvae infected with Ascosphaera apis. [Results]  The CDS of Am-caspase-3 is 1 023 bp in length and encodes 340 amino acids. The molecular formula of the Am-caspase-3 protein is C₁₇₆₀H₂₇₀₃N₄₅₉O₅₃₉S₂₃, and its relative molecular weight is 39 kD. There are 44 potential phosphorylation modification sites and no transmembrane structure or signal peptide region. Am-caspase-3 is mainly distributed in the cytoplasm. Its secondary structure is comprised of 184 random coils, 90 α-helices, 46 extended strands and 20 β-turns. One structural domain and ten conserved motifs were identified. There is 73.00% homology between Am-caspase-3 and its template 1m72.1.A. There is an interaction network among Am-caspase-3 and 12 other proteins such as CytC. There is 81.23% homology between the caspase-3 of A. mellifera and A. cerana, with the homologous genes of each species clustering within a single clade on the phylogenetic tree. Am-caspase-3 was differentially expressed in different worker tissues. Expression was significantly higher (P < 0.01) in the midgut than in the other 6 tissues tested, and significantly higher (P < 0.01) in 3-day-old larvae than in eggs, 7-day-old prepupae, 8-day-old prepupae or 12-day-old pupae. Expression of Am-caspase-3 in 2, 6, 15 and 18-day-old workers was significantly higher (P < 0.05) than in 1-day-old workers. Expression was significantly downregulated (P < 0.05) 1, 4, 7, 10 and 13 days, after infection with Nosema ceranae. Conversely, expression was significantly upregulated (P < 0.05) 1, 2, and 3 days, after infection with Ascosphaera apis. [Conclusion]  The CDS of Am-caspase-3 was successfully cloned. Am-caspase-3 is a putative acidic, hydrophilic, intracellular protein that interacts with 11 other proteins including CytC, Am-caspase-3 is most homologous to Apis cerana caspase-3. Am-caspase-3 potentially participates in the developmental processes of different tissues and developmental stages of A. mellifera workers, as well as in the immune response to infection by pathogenic fungi.