Heavy metal pollution is an increasing global concern. Among heavy metals, mercury (Hg) is especially dangerous because of its massive release into the environment and high toxicity, especially for aquatic organisms. The molecular response mechanisms of algae to Hg exposure are mostly unknown. Here, we combine physiological, biochemical, and transcriptomic analysis to provide, for the first time, a comprehensive view on the pathways activated in Chromera velia in response to toxic levels of Hg. Production of hydrogen peroxide and superoxide anion, two reactive oxygen species (ROS), showed opposite patterns in response to Hg²⁺ while reactive nitrogen species (RNS) levels did not change. A deep RNA sequencing analysis generated a total of 307,738,790 high-quality reads assembled in 122,874 transcripts, representing 89,853 unigenes successfully annotated in databases. Detailed analysis of the differently expressed genes corroborate the biochemical results observed in ROS production and suggests novel putative molecular mechanisms in the algal response to Hg²⁺. Moreover, we indicated that important transcription factor (TF) families associated with stress responses differentially expressed in C. velia cultures under Hg stress. Our study presents the first in-depth transcriptomic analysis of C. velia, focusing on the expression of genes involved in different detoxification defense systems in response to heavy metal stress.