Cardiovascular diseases remain the leading cause of mortality worldwide, with hypercholesterolemia being a major contributing factor. Marine-derived compounds, particularly from brown algae, have emerged as promising candidates for functional food development targeting lipid disorders. The main objective of this work was study the molecular effects of a purified aqueous extract of Fucus vesiculosus, rich in phlorotannins and peptides, on differentiated Caco-2 cells, simulating the human intestinal barrier. Using untargeted metabolomics (LC-HRMS/MS) and gel-based proteomics, it was explored the extract’s impact on cellular metabolism and protein expression. Metabolomic analysis revealed significant alterations in lipid-related metabolites, including increased levels of fatty acid amides and C16 phytosphingosine, compounds associated with anti-inflammatory and cholesterol absorption-modulating properties. Notably, a marked depletion of glutathione was observed, suggesting oxidative stress induction, which may have therapeutic implications in cancer contexts. Proteomic profiling identified several proteins exclusively expressed in extract-treated cells, including Niemann–Pick C1 (NPC1) and fatty acid-binding protein 1 (FABP1), both key regulators of cholesterol transport and homeostasis. Gene ontology enrichment highlighted biological processes related to intestinal absorption and lipid metabolism, reinforcing the extract’s potential role in modulating cholesterol uptake. This integrative omics approach provides novel insights into the intestinal-level mechanisms of F. vesiculosus bioactive compounds. The findings support its hypocholesterolemic potential described in traditional medicine and suggest additional anti-inflammatory and antitumor properties, positioning this seaweed extract as a promising candidate for nutraceutical development and cardiovascular disease prevention.