Our aim was to conduct a pilot case-control study of RNA expression profile using RNA sequencing of rectosigmoid mucosa of 9 females with irritable bowel syndrome-diarrhea [IBS-D] with accelerated colonic transit and 9 female healthy controls. Mucosal total RNA was isolated, purified, and next generation pair-end sequencing was performed using Illumina TruSeq. Analysis was carried out using: a targeted approach towards 12 genes previously associated with IBS, and a hypothesis-generating approach. Of the 12 targeted genes tested, patients with IBS-D had decreased mRNA expression of TNFSF15 (fold change controls to IBS-D: 1.53, p=0.01). Overall, up- and downregulated mRNA expressions of 21 genes (p values 10^-5 to 10^-8; p values with FDR shown) were potentially relevant to IBS-D: neurotransmitters [P2RY4 (p=0.001), VIP (p=0.02)]; cytokines [CCL20 (p=0.019)]; immune function [C4BPA complement cascade (p=0.0187)], and IFIT3 [interferon-related, p=0.016]; mucosal repair and cell adhesion [TFF1 (trefoil protein, p= 0.012)], RBP2 (retinol binding protein, p=0.017), FN1 (fibronectin, p=0.009)]; and ion channel functions [GUCA2B (guanylate cyclase, p= 0.017), PDZD3 (PDZ domain-containing protein 3, p=0.029)]. Ten genes associated with functions related to pathobiology of IBS-D were validated by RT-PCR: There was significant correlation in fold changes of the selected genes (Rs=0.73, p=0.013). Up- or downregulation of P2RY4, GUC2AB, RBP2, FNI and C4BPA genes were confirmed on RT-PCR, which also revealed upregulation of FXR and ASBT. RNA seq and RT-PCR analysis of rectosigmoid mucosa in IBS-D show transcriptome changes that provide the rationale for validation studies to explore the role of mucosal factors in the pathobiology of IBS-D.