Updated publication reference for DOI(s): http://dx.doi.org/10.1038/npjmgrav.2015.16. Background/Objectives: The waterflea Daphnia is an interesting candidate for biore- generative life support systems (BLSS). These animals are particularly promising be- cause of their central role in the limnic food web and its mode of reproduction. How- ever, 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 gener- ated 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). Substan- tial fractions of these proteins are involved in actin microfilament organisation, in- dicating the disruption of cytoskeletal structures during clinorotation. Furthermore, proteins involved in protein folding were identified, suggesting altered gravity in- duced break-down of protein structures in general. In addition, simulated micro- gravity 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 con- ditions 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.