Daphnia (Daphnia pulex) is a small planktonic crustacean and a key constituent of aquatic ecosystems. It is commonly used as a model organism for studying environmental toxic challenges. In the past decade, a Daphnia genomic information and proteomic dataset has been developed. This dataset has expanded the opportunity to relate toxicological effects with “Daphnia proteomics” as it integrates proteomic knowledge in Daphnia, those approach will provide greater insights for toxicological research. In order to exploit Daphnia for ecotoxicological research, information on the post-translational modification (PTM) of proteins is necessary as this is a critical regulator of biological processes. Acetylation of lysine (Kac) is a reversible and highly regulated PTM that is associated with diverse biological functions. However, a comprehensive description of Kac in Daphnia is not yet available. Here, to understand the cellular distribution of lysine acetylation in Daphnia, we identified 98 acetylation sites in 65 proteins by immunoprecipitation using an anti-acetyllysine antibody and an liquid chromatography system supported by mass spectroscopy. We identified 28 acetylated sites connected with metabolic proteins and 6 acetylated enzymes associated with the TCA cycle in Daphnia. From GO and KEGG enrichment analyses, we showed that Kac in D. pulex is highly enriched in proteins associated with metabolic processes. Our data provide the first global analysis of lysine acetylation in D. pulex. The expanded proteomic dataset will be an important resource for the functional analysis of Kac in D. pulex and it will be nice to have a first step done using a promising future model organism.