This study was performed to determine whether low frequency ultrasound (LFU) irradiation, Papaverine (PA) infusion and combination LFU irradiation with PA infusion opened the blood-tumor barrier (BTB) by affecting tight junctions (TJ)-associated proteins zonula occluden-1 (ZO-1), occludin and caludin-5. In a rat brain glioma model, we found that the mRNA and protein expression levels of ZO-1, occludin and claudin-5 were decreased by LFU irradiation and PA infusion. LFU-induced and PA-induced decrease of ZO-1, occludin and claudin-5 was further decreased after combining LFU irradiation with PA infusion. Immunohistochemistry assay showed that the decreased expression of ZO-1, occludin and claudin-5 was the most obvious in the tumor capillaries. Meanwhile, Evans blue assay showed that the permeability of BTB was increased, and transmission electron microscopy (TEM) indicated that TJ was opened. This led to the conclusion that LFU irradiation and PA infusion together can open the BTB by paracellular pathway. Significantly down-regulated expression levels of ZO-1, occludin and claudin-5 might be one of the molecular mechanisms of combining LFU and PA enhancing the permeability of BTB.