Microalgae are widely used as representative primary producers in ecotoxicology, while macrophytes are much less studied. Here we compared the bioavailability and cellular toxicity pathways of 2 h-exposure to 10−6 mol L−1 Cu in the macrophyte Elodea nuttallii and the green microalga Chlamydomonas reinhardtii. Uptake rate was similar but faster in the algae than in the macrophyte, while RNA-Sequencing revealed a similar number of regulated genes. Early-regulated genes were congruent with expected adverse outcome pathways for Cu with Gene Ontology terms including gene regulation, energy metabolism, transport, cell processes, stress, antioxidant metabolism and development. However, the gene regulation level was higher in E. nuttallii than in C. reinhardtii and several categories were more represented in the macrophyte than in the microalga. Moreover, several categories including oxidative pentose phosphate pathway (OPP), nitrate metabolism and metal handling were only found for E. nuttallii, whereas categories such as cell motility, polyamine metabolism, mitochondrial electron transport and tricarboxylic acid cycle (TCA) were unique to C. reinhardtii. These differences were attributed to morphological and metabolic differences and highlighted dissimilarities between a sessile and a mobile species. Our results highlight the efficiency of transcriptomics to assess early molecular responses in biota, and the importance of studying more aquatic plants for a better understanding on the impact and fate of environmental contaminants.