In recent years, the interest in the use of vitellogenin (VTG) as a biomarker of endocrine disruption in fish has led to VTG being considered as a potential tool in invertebrates. Among aquatic invertebrate models in ecotoxicology, the copepods are considered as reference species in marine, estuarine and freshwater ecosystems. In this context, we identified a VTG cDNA in Eurytemora affinis. The Ea-VTG2 cDNA is 5416bp in length with an open reading frame (ORF) of 5310bp that encodes a putative protein of 1769 amino acids residues. Phylogenetic analysis confirmed the hypothesis of a VTG duplication event before the emergence of the copepod species. The analysis of the Ea-VTG2 expression by qPCR in males and females according to their reproductive stages allowed transcript basal levels to be determined. The expression pattern revealed a gradual increase of transcript levels during maturation in females. Important inter-sex differences were observed with a VTG level in males ranging from about 1900- to 6800-fold lower than in females depending on their stage. Moreover, the protein was only detected in ovigerous females. The inducibility of Ea-VTG2 by chemicals was studied in males exposed to either a model of endocrine disruptor in vertebrates i.e. 4-nonylphenol (4-NP) or a crustacean hormone i.e. Methyl Farnesoate (MF), and in males sampled from a multi-contaminated estuary. No induction was highlighted. The VTG should not be considered as an appropriate biomarker in E. affinis as previously suggested for other crustaceans.