Chile is the second largest producer of salmonids in the world. This status has driven the industry to adopt increasingly intensive practices, which in turn expose fish to various stressors—both environmental and infectious—that negatively affect intestinal health, fish growth, and overall production efficiency. As a result, nutritional strategies including functional additives capable of modulating immune and antioxidant responses, as well as preserving epithelial integrity, are being sought out. One additive with great potential is Peruvian maca (Lepidium meyenii), an Andean tuber with well-described antioxidant, immunomodulatory, and adaptogenic properties in mammalian models, but whose application in fish remains poorly explored. Considering these facts, this study aimed to evaluate the cytoprotective, antioxidant, and immunomodulatory potential of an ethanolic extract of yellow maca in the RTgutGC intestinal cell line derived from rainbow trout. Cytotoxicity analysis revealed no signs of cell damage or alterations at extract concentrations between 1 and 25 µg/mL. At the gene expression level, treatments with 5, 10, and 15 µg/mL of extract led to a decrease in tnf-α mRNA abundance and an increase in tgf-β, as well as a transient increase in catalase (cat) and glutathione reductase (gr) transcripts at 6 h, followed by a reduction at 24 h. An increase in zonula occludens 1 (zo-1) transcript levels was also observed, suggesting anti-inflammatory, antioxidant, and reinforcement of epithelial barrier function. Moreover, pretreatment of RTgutGC cells with 5 µg/mL maca extract counteracted the LPS-induced increase in tnf-α and il-8 transcripts and the reduction in tgf-β abundance (LPS 50 µg/mL). In parallel, cells pretreated with maca and later exposed to H₂O₂ (250 µM) showed significantly lower levels of cat and gr transcripts, indicating moderated oxidative stress responses. Finally, functional assays in RTgutGC cells cultured in transwell systems and pretreated with maca showed attenuation of the H₂O₂-induced drop in transepithelial electrical resistance (TEER). Under the same conditions, a reduction in epithelial permeability was observed through decreased paracellular flux of FITC-dextran. These findings strongly support the anti-inflammatory, antioxidant, and barrier-stabilizing actions of maca extract on intestinal epithelial cell function, highlighting the potential of Lepidium meyenii as a functional feed additive to promote intestinal health in salmonids.