Contamination of freshwaters is increasing globally, with algae considered one of the most sensitive taxa to metal pollution. 72 h bioassays were used to explore the biochemical effects of copper (Cu) on the amino acid (AA) profile and proteome of Chlorella sp.. The Cu concentrations required to inhibit algal growth rate by 10 % (EC10) and 50 % (EC50) were 1.0 (0.7-1.2) µg L-1 and 2.0 (1.9-2.4) µg L-1, respectively. The AA profile of the algae showed increases in glycine and decreases in isoleucine, leucine, valine, and arginine, with increasing Cu. Cu was shown to influence the regulation of several proteins involved in energy production, including: photosynthesis, carbon fixation, glycolysis, and oxidative phosphorylation, which likely assists in meeting the increased energy demands of Chlorella when exposed to Cu. Cu exposure caused the up-regulation of proteins related to cellular processes and signalling, and the down-regulation of proteins related to ribosomal structure and protein translation. Changes in biomolecular pathways had direct effects on the AA profile of the algae and total protein content. This study shows the complex mode of action of Cu on Chlorella under environmentally realistic Cu concentrations and highlights several potential biomarkers for future investigation.