[Objective] This study aimed to reveal miRNA-mediated mechanism underlying Ascosphaera apis infection of Apis cerana cerana larvae. [Methods] Small RNA (sRNA) dataset of A. apis during infection (AaT) was screened out from sRNA-seq data from Ascosphaera apis-infected A. c. cerana 6-day-old larval guts. The filtered sRNA datasets from the purified spores (AaCK) and AaT were aligned against miRBase using Blast, followed by analyses of number and structural characteristics of pathogen miRNAs before and after Ascosphaera apis infection. Prediction, GO categorization and KEGG pathway enrichment analysis of targets of DEmiRNAs were conducted using related software. The regulation network between DEmiRNAs and corresponding targets was visualized using Cytoscape. Stem-loop RT-PCR, qPCR and molecular cloning were used to verify the reliability of our sequencing data. [Results] Totally, 380 and 387 miRNAs were identified in AaCK and AaT, respectively. The length of Ascosphaera apis miRNAs were mainly distributed between 18 nt and 25 nt; and the first base had a U bias. There were 155 up-regulated and 115 down-regulated miRNAs in AaCK vs AaT, targeting 6091 and 6145 mRNAs. Targets of DEmiRNAs were involved in 15 biological processes, 12 cell components and 11 molecular functions. Additionally, these targets were engaged in 123 pathways, regulating material metabolisms, energy metabolisms and signaling pathways. Moreover, complex regulation networks existed between DEmiRNA and corresponding targets, among them miR-29-x, miR-250-x, miR-4968-y, miR-11200-x, novel-m0023-5p, novel-m0130-5p and novel-m0135-5p can target mRNAs associated with cysteine proteinase, DNA methyltransferases and chitinase; miR-7-x, miR-9-z, miR-319-y and miR-5951-y can simultaneously regulate MAPK signaling pathway; miR-250-x may be involved in cross-kingdom regulation between A. apis and A. c. cerana larvae. [Conclusion] Our results revealed DEmiRNAs may participate in the infection process of A. apis via regulating targets associated with material and energy metabolisms, pathogen proliferation, virulence, and several signaling pathways; several key miRNAs including miR-7-x were potential targets for chalkbrood control.