Background/Objectives: The waterflea Daphnia is an interesting candidate for bioregenerative life support systems (BLSS). These animals are particularly promising because of their central role in the limnic food web and its mode of reproduction. However the response of Daphnia to altered gravity conditions has to be investigated especially on the molecular level to evaluate the suitability of Daphnia for BLSS in space. Methods: In this study we applied a proteomic approach to identify key proteins and pathways involved in the response of Daphnia to simulated microgravity generated by a 2D-clinostat. We analysed 5 biological replicates using 2D-DIGE proteomic analysis. Results: We identified 109 protein spots differing in intensity (p < 0.05). Substantial fractions of these proteins are involved in actin microfilament organisation indicating the disruption of cytoskeletal structures during clinorotation. Furthermore proteins involved in protein folding were identified suggesting altered gravity induced breakdown of protein structures in general. In addition simulated microgravity increased the abundance of energy metabolism related proteins indicating an enhanced energy demand of Daphnia. Conclusion: The affected biological processes were also described in other studies using different organisms and systems either aiming to simulate microgravity conditions or providing real microgravity conditions. Moreover most of the Daphnia protein sequences are well conserved throughout taxa indicating that the response to altered gravity conditions in Daphnia follows a general concept.