Blastocystis spp. is a common intestinal parasite in humans and animals that has been associated with acute or chronic digestive disorders such as irritable bowel syndrome. Serotonin mediates intestinal motility, sensation, and secretory function in the normal intestinal tract. Serotonin (5-HT) signaling is decreased in animal models of colitis, as well as the colonic mucosa of humans with ulcerative colitis and irritable bowel syndrome. Altered 5-HT signaling may underlie the abnormal motility, secretion, and sensation seen in these inflammatory gut disorders. Molecular recognition of pathogens is facilitated through Toll-like receptors (TLR). The colonic epithelium expresses relatively high concentrations of mRNA for TLR2 and TLR4. Little is known about the expression patterns for these pattern recognition receptors in response to Blastocystis spp. infection in the human colon. The human colonic epithelial cell line, CACO-2 was used to examine whether enteric 5-HT signaling, TLR2 and TLR4 expression are altered in CACO – 2 intestinal cells in the presence of Blastocystis sp. ST1. Messenger RNA concentrations of components of serotonin signaling (Serotonin transporter, Tryptophan Hydroxylase – TPH1), TLR2, and TLR4 were quantified with a quantitative real-time reverse transcription polymerase chain reaction in CACO – 2 cells that had been co-cultured for 24 hours with live Blastocystis sp. ST1. Protein transcription was analyzed with a Western Blot technique. Live Blastocystis sp. ST1 parasites inhibited messenger RNA expression of serotonin transporter in CACO-2 cells and had no effect on TPH1 messenger RNA expression. Live Blastocystis sp. ST1 parasites alone did not activate TLR-2 or TLR-4. This study evaluated the effect of Blastocystis sp. ST1 on serotonergic signaling and TLR activation using an in vitro tissue culture model.