[Objectives]  To investigate the morphological effects of infection of honeybees by Nosema ceranae, including the main tissues infected, the effects on protein expression in the midgut and the regulation of the honeybee immune response. [Methods]  Total honeybee midgut proteins were extracted and screened using SDS-PAGE and differentially expressed proteins identified using LC-MS/MS. Differential expression of proteins was verified with semi-quantitative, and quantitative, RT-PCR. [Results]  There was no significant difference in the body weight of infected and uninfected (control) Apis cerana cerana Fabricius or Apis mellifera Ligustica. However, there were significant differences in the intestinal morphology of infected and uninfected bees. Furthermore, two differentially expressed midgut protein bands were identified in A. mellifera and A. cerana cerana. Thirty-five candidate proteins were obtained by MS in A. mellifera and 11 in A. cerana cerana. Four days after infection, expression levels of arginine kinase (AK) and glycerol triphosphate dehydrogenase (GPDH) in A. mellifera, were up-regulated 2.02 times, 2.21 times, respectively, relative to the control group, whereas thioredoxin reductase (TrxR) and arginine in A. cerana cerana were up-regulated 1.72 times, and 1.88 times, respectively. All data were consistent with the results of SDS-PAGE analysis. [Conclusion]  The expression of proteins related to basic metabolism, immune response, oxidative stress and energy metabolism in the honeybee midgut (such as AK and GPDH in A. mellifera and AK and TrxR in A. cerana cerana) were significantly up-regulated after infection by N. ceranae, suggesting that these proteins may be involved in the immune defense of intestinal tissues. This study provides a rich data base for in-depth study of the immune and metabolic responses of the honeybee midgut to infection by N. ceranae.