Mercury can be very toxic at low environmental concentrations by impairing immunological, neurological, and other vital pathways in humans and animals. Aquatic ecosystems are heavily impacted by mercury pollution, with evidence of biomagnification through the food web. We examined the effect of mercury toxicity on the spleen, one of the primary immune organs in fish, in natural populations of the three-spined stickleback ( Gasterosteus aculeatus Linnaeus, 1758). Our aim was to better understand adaptation to high mercury environments by investigating transcriptomic changes in the spleen. Three stickleback populations with mean Hg muscle concentrations above and three populations with mean Hg muscle concentrations below the European Biota Quality Standard of 20 ng/g wet weight were selected from the Scheldt and Meuse basin in Belgium. We then conducted RNA sequencing of the spleen tissue of 22 females from these populations. We identified 136 differentially expressed genes between individuals from populations with high and low mean mercury content. The 129 genes that were upregulated were related to the neurological system, immunological activity, hormonal regulation, and inorganic cation transporter activity. Seven genes were downregulated and were all involved in pre-mRNA splicing. The results are indicative of our ability to detect molecular alterations in natural populations that exceed an important environmental quality standard. This allows us to assess the biological relevance of such standards, offering an opportunity to better describe and manage mercury-associated environmental health risks in aquatic populations.