The clinical chemotherapy of brain tumors has been limited by the blood-tumor barrier (BTB). Low-frequency ultrasound (LFU) in combination with microbubbles might be a useful method for local drug delivery. However, the underlying mechanism remains unclear. In this study, we asked whether LFU changed the permeability of BTB by regulating the tight junction-related proteins. The permeability of BTB was evaluated by Evans blue dye, and the protein and mRNA expression levels of tight junction-related proteins claudin-5, occludin, and ZO-1 were determined by immunohistochemical staining, RT-PCR, and western blot assays. We found that the permeability of BTB increased significantly after LFU exposure in the presence of Optison. The mRNA and protein expression levels of claudin-5, occludin, and ZO-1 decreased significantly at 3 h, restored gradually and nearly recovered after 12 h. The correlation between the increase of BTB permeability and the reduction of tight junction-related proteins suggests that LFU combined with microbubbles may be involved in the opening of the BTB by the tight junction-related proteins.