The effect of hydrostatic pressure on the paracellular ion conductance (Gp) composed of the Na+ conductance (GNa) and the Cl− conductance (GCl) has been Investigated. Gp, GNa and GCl were time-dependently increased after applying an osmotic gradient generated by NaCl with basolateral hypotonicity. Hydrostatic pressure (1–4 cm H2O) applied from the basolateral side enhanced the osmotic gradient-induced increase in Gp, GNa and GCl in a magnitude-dependent manner, while the hydrostatic pressure applied from the apical side diminished the osmotic gradient-induced increase in Gp, GNa and GCl. How the hydrostatic pressure influences Gp, GNa and GCl under an isosmotic condition was also investigated. Gp, GNa and GCl were stably constant under a condition with basolateral application of sucrose canceling the NaCl-generated osmotic gradient (an isotonic condition). Even under this stable condition, the basolaterally applied hydrostatic pressure drastically elevated Gp, GNa and GCl, while apically applied hydrostatic pressure had little effect on Gp, GNa or GCl. Taken together, these observations suggest that certain factors controlled by the basolateral osmolality and the basolaterally applied hydrostatic pressure mainly regulate the Gp, GNa and GCl.