Intensive aquaculture faces challenges such as infectious diseases, environmental stress, and impaired immune function. Consequently, natural feed additives are being explored as potential alternatives to conventional interventions. Montmorillonite, a smectite clay mineral, has attracted attention because of its adsorptive properties and cation-exchange capacity. This study evaluated the effects of dietary montmorillonite on a non-specific immune-related response, the measured serum lysozyme-related parameter, and behavioral stress in juvenile rainbow trout (Oncorhynchus mykiss). A total of 450 fish were assigned to three dietary treatments with three replicates each: a control diet, a diet containing 2% montmorillonite, and a diet containing 4% montmorillonite, for 56 days. Water quality, the serum lysozyme-related response, final survival rate, and behavioral stress score were assessed. Water quality remained within the expected range throughout the experimental period. Dietary montmorillonite significantly increased the diameter of the bacterial growth-inhibition zone in the Lysoplate assay, which was used as the measured indicator related to serum lysozyme activity (P < 0.05). In addition, the behavioral stress score was significantly lower in fish receiving 4% montmorillonite than in the control group, whereas no significant differences were detected in final survival rate among treatments. These findings indicate that dietary montmorillonite supplementation was associated with changes in the measured serum lysozyme-related response and a reduction in behavioral stress scores under the conditions of the present study. Further studies are required to determine its effects on growth performance, feed efficiency, physiological mechanisms, and long-term health outcomes.