We aim to evaluate the effects of four Nosema spores’ isolates, (i) and (ii) N. ceranae isolated from A. mellifera hosts from two different geographical origins, (iii) N. ceranae from A. cerana host and (iv) N. apis from A. mellifera, on the A. mellifera on gut proteomics at the early stage of infection. To dissect the molecular mechanism responsible of the susceptibility of A. mellifera to Nosema, we investigated by high-resolution proteomics (LC-ESI-MS/MS) and differential label-free quantification of proteins (LFQ) the molecular cross-talk existing between different species and isolates of N. apis and N. ceranae, and the targetted gut tissue of A. mellifera. To reach the objectives of this study, we performed a bottom-up proteomic analysis on the different anatomical sections of the gut tissue (esophagus, crop, midgut, ileum and rectum) at an early stage of the exposition to Nosema spores (4 days). Then, we focused on the midgut, the region targeted by Nosema sposres for germination and, as we found out, the second region with the highest load of Nosema proteins, after the rectum, to perform differential quantitative proteomic analyses and acquire series of up- and down-regulated proteins. We discussed the different pathways observed to be impacted by different Nosema species and isolates with a main focus on the deregulated metabolic and response to stimuli processes.