The MIX formulation (Carob, B. longum GA24, ribonucleotides) enhanced intestinal barrier integrity by boosting Caco-2 viability, tight junctions, butyrate synthesis, and reducing ROS. In the gut testis co-culture, MIX protected Sertoli cells against oxidative stress, improving viability, ATP/NO production, and suppressing apoptotic and inflammatory markers. It further promoted Sertoli cell maturation and hormonal regulation, markedly increasing AR, p27, SGP-2, testosterone, and FSHR expression, thereby supporting spermatogenesis via the gut testis axis.