[Objectives]  Ascosphaera apis specifically infects honey bee larvae leading to chalkbrood disease, resulting in a serious decline in honey bee numbers and the quantity of honey bee products such as honey.  High-throughput sequencing technology has not, so far, been used to study chalkbrood. In order to reveal the molecular mechanism regulating the responses of western honey bee larvae to A. apis, we used RNA sequencing to sequence the guts of normal and A. apis-challenged 4-day-old larvae of Apis mellifera ligustica. [Methods]  Guts of an AmCK (un-treated group) and AmT (A. apis-treated group) were sequenced on an Illumina Hiseq 2500 (PE125) platform and the raw data were filtered with Perl script. Sequencing saturation analysis and Pearson correlation coefficients were calculated using Program R software. Mapping of reads to the reference genome of A. mellifera was conducted using SOAP aligner/soap2 software and GO classification and KEGG pathway enrichment analysis were carried out based on the GO and KEGG databases. [Results]  181 096 194 raw reads, and after filtration, 179 078 764 clean reads, were obtained from RNA-seq. The results of differentially expressed gene (DEG) analysis showed that the number of up- and down-regulated genes were 4 and 20, respectively. Gene ontology (GO) classification suggests these DEGs are grouped into 19 GO terms, and that among them, only several genes assigned to binding and catalytic activity were up-regulated. KEGG pathway enrichment analysis indicated that 1 and 1 DEG were enriched in ribosome and oxidative phosphorylation, respectively. [Conclusion]  Findings in the present study not only reveal larval responses of A. m. ligustica to A. apis during the early stage of infection, but also lay a foundation for illustration of the molecular mechanisms underlying host responses to A. apis.