Sec61β is the β subunit of the Sec61 translocon and is responsible for expression and delivery of basolateral membrane proteins, including claudins, major constituents of tight junction (TJ). In the present study, the effect of Sec61β overexpression on TJ barrier functions in Madin-Darby canine kidney (MDCK) cells were investigated by monitoring transepithelial electrical resistance (TER) and the expression and distribution of claudins. We adopted the time required by TER to reach 50% (T1/2) as a measure of TJ modulation rate. Sec61β overexpression increased TER by post-transcriptionally upregulating claudin-4 expression and resulted in increased TER. Sec61β overexpression increased TJ modulation rates (lower T1/2), in conjunction with enhanced delivery of claudin-4 from and to plasma membranes. Marked co-distribution and indirect association of claudin-4 with Sec61β were observed, contributing to the enhanced delivery of claudin-4. Thus, Sec61β may be a novel TJ modulation target, including barrier function and modulation rates for drug delivery systems.