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Over recent decades, Northern Patagonia in Chile has seen significant growth in agriculture, livestock, forestry, and aquaculture, disrupting lake ecosystems and threatening native species. These environmental changes offer a chance to explore how anthropization impacts zooplankton communities from a molecular-ecological perspective. This study assessed the anthropogenic impact on Daphnia pulex by comparing its proteomes from two lakes: Llanquihue (anthropized) and Icalma (oligotrophic). Results showed substantial differences in protein expression, with 17 proteins upregulated and 181 downregulated in Llanquihue, linked to elevated levels of copper, manganese, dissolved solids, phosphate, and nitrogen. These stressors caused metabolic damage and environmental stress in D. pulex . Our findings highlight the importance of monitoring pollution's effects on Northern Patagonian ecosystems, especially on keystone species like D. pulex , essential for ecosystem stability. This research provides fresh molecular-ecological insights into pollution's impacts, a perspective rarely addressed in this region. Understanding these effects is critical for conserving natural resources and offers pathways to study adaptive mechanisms in keystone species facing pollution. This approach also informs strategies for ecosystem management and restoration, addressing both immediate and long-term challenges in Northern Patagonian aquatic environments.