DA-6886 is a novel serotonin (5-hydroxytrypamine [5-HT]) receptor 4 agonist for the potential treatment of constipation-predominant irritable bowel syndrome. The purpose of this study was to validate the quantitative assay of DA-6886 in rat plasma and to evaluate the pharmacokinetics and tissue distribution of DA-6886 in rats. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the robust quantification of DA-6886 in rat plasma was successfully validated and applied to the pharmacokinetic studies in rats. The pharmacokinetic parameters of DA-6886 in rats were evaluated following single intravenous or oral administration at three dose levels (2, 10, and 20 mg/kg). DA-6886 exhibited a smaller dose-normalized area under the plasma concentration-time curve (AUC) values and faster clearances in the low-dose group than in the high-dose group following both intravenous and oral administration. The steady-state volume of distribution (Vss) of DA-6886 was relatively large (4.91-7.84 L/kg), which was consistent with its high distribution to the liver, kidney, lung, and digestive tract, and was dose-independent. After oral administration, the extent of absolute bioavailability (F) tended to increase (18.9-55.0%) with an increasing dose. The slope of the log-transformed AUC and/or Cmax values versus log dose was greater than unity and greater for oral administration (~1.9) than for intravenous administration (~1.1). Because the nonlinear pharmacokinetics of DA-6886 was more obviously observed after oral administration, it appears that the saturation of pre-systemic intestinal and/or hepatic first-pass extraction of DA-6886 at high doses occurred.