The effect of Wellsolve, a new solubilizing agent, on the function of intestinal membrane barrier and transporters including P-glycoprotein (P-gp) and peptide transporter (PEPT1) was examined by an in vitro diffusion chamber and an in situ closed loop method. The model drugs used in this study were 5(6)-carboxyfluorescein (CF), rhodamine123 (a P-glycoprotein substrate), cephalexin (a typical substrate for PEPT1) and griseofulvin (a BCS Class II drug). Intestinal absorption of CF was not affected by the addition of 1—10% (v/v) Wellsolve, while 20% (v/v) Wellsolve significantly enhanced its intestinal absorption by the in situ absorption study. Therefore, this finding suggested that high concentration of Wellsolve might alter the intestinal barrier function. The mucosal to serosal (absorptive) and serosal to mucosal (secretory) transport of rhodamine123 was significantly inhibited in the presence of 5.0—20% (v/v) of Wellsolve, suggesting that Wellsolve might not affect the function of P-gp in the intestine. The intestinal transport of cephalexin was not affected in the presence of Wellsolve, suggesting that this solubilizing agent might not change the function of PEPT1 in the intestine. In the toxicity studies, we found that 1—10% (v/v) Wellsolve did not change the release of lactate hydrogenase (LDH) and protein from the intestinal membranes. Furthermore, intestinal absorption of griseofulvin in the presence of 10% (v/v) Wellsolve significantly increased as compared with the control. In summary, Wellsolve at lower concentrations might be a potent and safe solubilizing agent for improving the solubility and absorption of poorly water-soluble drugs including griseofulvin.