The laxative effects of aqueous extract of Liriope platyphylla (AEtLP) on loperamide (Lop)-induced constipation have been reported; however, the key compounds and the mechanism underlying these effects remain unclear. Therefore, the laxative effects of five candidates derived from L. platyphylla: Diosgenin (DG), 5-hydroxymethylfurfural (5-HMF), adenosine (AD), hydroxypropyl cellulose (HPC) and uridine (UD) were investigated by examining the alteration of G protein a (G alpha) expression, protein kinase C (PKC) phosphorylation and inositol triphosphate (IP3) concentration levels in the 5-hydroxytryptamine (5-HT; serotonin) receptor signaling pathway. Primary rat intestine smooth muscle cells (pRISMCs), intestinal epithelial cells (IEC)-18 and B35 cells were cotreated with Lop and the five compounds in order to screen the candidates. AEtLP, prucalopride (PCP) and bisacodyl (BS) served as positive controls. In pRISMCs, G alpha expression levels were recovered in the majority of candidate -treated groups, whereas PKC phosphorylation recovery was observed only in the DG, 5-HMF and AD treatment groups. In IEC-18 cells, the AD treatment group mimicked the effects of PCP on PKC phosphorylation levels, whereas the DG, 5-HMF, HPC and UD treatment groups mimicked the effects of AEtLP and BS. In B35 cells, a greater upregulation of PKC phosphorylation levels were observed in the UD treatment group compared with the PCP and BS treatment groups, whereas DG, 5-HMF and AD treatment reduced the PKC phosphorylation levels to a greater extent than AEtLP treatment. However, effects similar to AEtLP, PCP and BS on G alpha expression levels were not detected in any treatment groups in IEC-18 and B35 cells. Furthermore, the level of IP3 was enhanced only in pRISMCs, in which all five candidates were effective, while the greatest concentration was observed in the UD treatment group. In conclusion, the results of the present study suggest that UD may be considered the compound with the greatest laxative activity, which may regulate the 5-HT receptor signaling pathway.