Agri-food industries generate several by-products, including protein-rich materials currently treated as waste. Lupine species could be a sustainable alternative source of protein compared to other crops such as soybean or chickpea. Protein hydrolysates contain bioactive peptides that may act positively in disease prevention or treatment. Inflammatory responses and oxidative stress underlie many chronic pathologies and natural treatment approaches have gained attention as an alternative to synthetic pharmaceuticals. Recent studies have shown that lupin protein hydrolysates (LPHs) could be an important source of biopeptides, especially since they demonstrate anti-inflammatory properties. However, due to their possible degradation by digestive and brush-border enzymes, it is not clear whether these peptides can resist intestinal absorption and reach the bloodstream, where they may exert their biological effects. In this work, the in vitro cellular uptake/transport and the anti-inflammatory and antioxidant properties of LPH were investigated in a co-culture system with intestinal epithelial Caco-2 cells and THP-1-derived macrophages. The results indicate that the LPH crosses the human intestinal Caco-2 monolayer and exerts anti-inflammatory activity in macrophages located in the basement area by decreasing mRNA levels and the production of pro-inflammatory cytokines. A remarkable reduction in nitric oxide and ROS in the cell-based system by peptides from LPH was also demonstrated. Our preliminary results point to underexplored protein hydrolysates from food production industries as a novel, natural source of high-value-added biopeptides.