[Objectives]  To analyze microRNAs (miRNAs) and their target genes in the the honeybee Apis mellifera ligustica larval gut, and organ that is the main site of food digestion and nutrient absorption and that is also important for pathogen resistance, and reveal the role of miRNAs in honeybee development and growth. [Methods]  The guts of 4, 5 and 6-day-old larvae were sequenced using small RNA sequencing (sRNA-seq) technology, followed by bioinformatic prediction and analysis of miRNAs. Target genes of DEmiRNAs were predicted with TargetFinder and annotated in the GO and KEGG databases with BLAST. Regulatory networks between DEmiRNAs and target mRNAs were constructed using Cytoscape. [Results]  A total of 96 329 456 clean tags were obtained from deep sequencing of larval gut samples and 560 miRNAs, including 45 novel miRNAs, were predicted. The length of these miRNAs was mainly between 17 and 27 nt, and the first base bias of miRNAs of different lengths varied significantly. Expression clustering showed that 331 miRNAs were shared in 4-, 5- and 6-day-old larvae. Although their expression levels were generally stable, the expression of specific miRNAs showed a general upward trend with larval development. TargetFinder software predicted 16 479 target genes of these miRNAs, of which 8 132 and 3 361 could be annotated in the GO and KEGG databases, respectively. Further investigation demonstrated that 224 target genes were enriched in the Wnt signaling pathway, and that 2 and 27 target genes were closely related to juvenile hormone and ecdysteroid regulation. [Conclusion]  These results provide a foundation for improving understanding of the molecular mechanisms underlying the larval development of A. m. ligustica.