Objective To investigate the regulation of blood-testis barrier by Rab13-PKA pathway in rats. Method First, shRNA vector targeting at Rab13 was constructed and then the Rab13 shRNA was transfected into the rat testis by injection. Western blot was used to detect the knock-down effect of Rab13 and the expression of blood-testis barrier (BTB) constituent proteins. PKA activity was detected by autoradiography and scintillation counting. Further, immunofluorescence analysis and phalloidin staining were applied to observe the distribution of occludin and F-actin, respectively. Results The expression level of Rab13 in the testis was reduced by approximately 70% after transfection of Rab13 shRNA as compared with the non-targeted control group (P<0.01), while the expression of BTB constituent proteins remained unchanged. PKA activity was significantly increased after Rab13 RNAi transfection (P<0.01). The distribution of occludin at BTB was remarkably increased after Rab13 RNAi silencing around stage VIII but not at other stages of the seminiferous epithelial cycle. The assembly of F-actin at BTB was also intensified in Rab13-silenced testis. Both the changes of distribution of occludin and F-actin induced by Rab13 shRNA were found to be antagonized by the PKA specific inhibitor H89. Conclusions Rab13 can modulate the distribution of occludin and F-actin at the blood-testis barrier in rats by regulating PKA activity, which may participate in the regulation of BTB function.