Honeybees are pivotal pollinators of crops and wild flora, and of great importance in supporting critical ecosystem balance. Nosema ceranae, a unicellular fungal parasite that infects midgut epithelial cells of honeybees, can dramatically reduce honeybee population and productivity. Here, midguts of Apis mellifera ligustica workers at 7 d and 10 d post inoculation (dpi) with sucrose solution (Ac7CK and Ac10CK) and midguts at 7 dpi and 10 dpi with sucrose solution containing N. ceranae spores (Ac7T and Ac10T) were sequenced using strand-specific cDNA library construction and next-generation sequencing. A total of 1956129858 raw reads were gained in this article, and after quality control, 1946489304 high-quality clean reads with a mean Q30 of 93.82% were obtained. The rRNA-removed clean reads were then aligned to the reference genome of Apis mellifera with TopHat2. For more insight please see “Genome-wide identification of long non-coding RNAs and their regulatory networks involved in Apis mellifera ligustica response to Nosema ceranae infection” [1]. Raw data were deposited in NCBI Sequence Read Archive (SRA) database under the BioProject number PRJNA406998. These data can be used for comparative analysis to identify differentially expressed coding RNAs and non-coding RNAs involved in host responses to N. ceranae stress, and for investigation of molecular mechanisms regulating N. ceranae-response.