Administration of oxygen is one of the most common interventions in medicine. Previous research showed that differential regulated proteins could be linked to hyperoxia-associated signaling cascades in different tissues. However, it still remains unclear which signaling pathways are activated by hyperoxia. The present study analyses hyperoxia-induced protein alterations in lung, brain, and kidney tissue using a proteomic and bioinformatic approach. Pooled data of 36 Wistar rats exposed to hyperoxia were used. To identify possible hyperoxia biomarkers, and to evaluate the relationship between protein alterations in hyperoxia affected organs and blood, proteomics data from brain, lung, and kidney were analyzed. Functional network analyses (IPA®, PathwaysStudio®, and GENEmania®) in combination with hierarchical cluster analysis (Perseus®) was used to identify relevant pathways and key proteins. Data of 54 2D-gels with more than 2500 significantly regulated spots per gel were collected. Thirty-eight differentially expressed proteins were identified and consecutively analyzed by bioinformatic methods. Most differences between hyperoxia and normoxia (21 proteins up-regulated, 17 proteins down-regulated) were found immediately after hyperoxia (15 protein spots), followed by day 3 (13 spots), and day 7 (10 spots). A highly significant association with inflammation and the inflammatory response was found. Cell proliferation, oxidative stress, apoptosis and cell death as well as cellular functions were revealed to be affected. Three hours of hyperoxia resulted in significant alterations of protein expression in different organs (brain, lung, kidney) up to seven days after exposure. Further studies are required to interpret the relevance of protein alterations in signaling cascades during/after hyperoxia.