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Objectives: To investigate the effects of radiation on paracellular pathway of rat submandibular glands (SMGs) and the mechanism of increasing secretion following treatment with pilocarpine. Materials and Methods: In situ irradiation models of SMGs in Wistar rats were conducted, and the glands were exposed to X‐radiation at a single dose of 20 Gy. Pilocarpine was intraperitoneally injected 60 min prior to radiation and continuous 6 days postirradiation for a total of 7 days. Salivary secretion, histological changes, pro‐inflammatory cytokines, alterations in tight junctions (TJs), and functional membrane proteins aquaporin‐5 (AQP5) and claudin‐4 mediated by the muscarinic acetylcholine M3 subtype receptor were determined at 1 and 12 weeks after irradiation. Results: Salivary secretion of the irradiated glands was reduced at 1 and 12 weeks. As well, acinar cell numbers, TJ width, and the levels of M3 receptor and AQP5 were decreased. In contrast, tumor necrosis factor‐α, interleukin 6, interleukin 1α, and the expression of the TJ protein claudin‐4 were significantly increased in irradiated SMGs. Notably, all the alterations were attenuated by pilocarpine treatment. Conclusions: Pilocarpine could improve the secretory function of irradiated rat SMGs via reducing inflammation, ameliorating the structural injury of TJs, and attenuating the up‐regulation of claudin‐4 expression. [ABSTRACT FROM AUTHOR]