Previous in vivo studies suggest that constitutive nitric oxide (cNO) can regulate Na- glucose co-transport (SGLT1) and Na:H exchange (NHE3) in rabbit intestinal villus cells. Whether these two primary Na absorbing pathways are directly regulated by cNO and the mechanisms of this regulation in the enterocyte is not known. Thus, non transformed rat small intestinal epithelial cell (IEC-18) were treated with L-NAME which directly decreased cNO in these cells. L-NAME treatment decreased SGLT1 in IEC-18 cells. Kinetic studies demonstrated that the mechanism of inhibition was secondary to a decrease in the affinity of the co-transporter for glucose without a change in the number of co-transporters. In contrast L-NAME treatment increased NHE3 in IEC-18 cells. Kinetic studies demonstrated that the mechanism of stimulation was by increasing the number of the exchangers without a change in the affinity for Na. RTQ-PCR and Western blot analysis of SGLT1 demonstrated no change in mRNA and protein respectively. RTQ-PCR and Western blot analysis of NHE3 indicated that NHE3 was increased by L-NAME treatment by an increase in mRNA and protein respectively. These results indicate that decreased cNO levels directly mediate the inhibition of SGLT1 and stimulation of NHE3 in intestinal epithelial cells. Thus, cNO directly but uniquely regulates the two primary Na absorptive pathways in the mammalian small intestine.