There are limited data on the influence of fructose rich diet on the male reproductive system. Kefir may have health beneficial effects, but its mechanism of action remains mostly unclear. Herein, we investigated the impact of dietary high fructose on tight junction proteins and mitogenic pathways in rat testis as well as their modulation by kefir supplementation. Twenty-two male Wistar rats (4 weeks old) were divided into the following three groups: Control; Fructose; Fructose + Kefir. Fructose was added to drinking water at concentration of 20% and administered to the rats for 15 weeks and kefir was supplemented by gavage once a day during final 6 weeks. Dietary fructose-induced testicular degeneration was associated with the downregulation of the blood-testis barrier proteins, claudin-11 and N-cadherin as well as SIRT1 expression in testicular tissue of rats. However, p38MAPK, p-p38MAPK and p-ERK1/2 levels were increased in testis of fructose-fed rats. Interestingly, JNK1 and p-JNK1 protein levels were decreased following this dietary intervention. Raf1, ERK1/2, and caspase 3 and TUNEL staining of the testis reveal the activation of apoptosis due to fructose intake. Kefir supplementation markedly promoted the expression of claudin-11, SIRT1, JNK1 and p-JNK1 but suppressed testicular mitogenic and apoptotic factors in fructose-fed rats.