Recombinant adeno-associated virus (rAAV) is the leading vector for gene replacement therapy; however, the roles and regulation of host proteins in rAAV production remain incompletely understood. In this comparative proteomic analysis, we focused on proteins in the nucleus, the epicenter of DNA uptake, transcription, capsid assembly, and packaging. HEK-293 cells were analyzed under the following three conditions: (i) untransfected, (ii) mock-transfected with the ITR and an unrelated plasmid, and (iii) triple-transfected with rAAV2 production plasmids. Cells were harvested at 24 and 72 h post-transfection, and nuclear fractions were processed using filter-aided sample preparation (FASP) followed by nano-scale liquid chromatography-tandem mass spectrometry (nLC-Orbitrap MS/MS). Across all samples, we identified 3384 proteins, revealing significant regulatory changes associated with transfection and rAAV production. Transfection alone accounted for some of the most substantial proteomic shifts, while rAAV production induced diverse regulatory changes linked to cell cycle control, structure, and metabolism. STRING analysis of significantly regulated proteins also identified an enrichment of those associated with the Gene Ontology (GO) term 'response to virus'. Additionally, we examined proteins with reported relation to adenoviral components. Our findings help to unravel the complexity of rAAV production, identify interesting targets for further investigation, and may contribute to improving rAAV yield.