Inhibition of constitutive nitric oxide (cNO) production inhibits SGLT1 activity by a reduction in the affinity for glucose without a change in Vmax in intestinal epithelial cells (IEC-18). Thus, we studied the intracellular pathway responsible for the posttranslational modification/s of SGLT1. NO is known to mediate its effects via cGMP which is diminished tenfold in L-NAME treated cells. Inhibition of cGMP production at the level of guanylyl cyclase or inhibition of protein kinase G also showed reduced SGLT1 activity demonstrating the involvement of PKG pathway in the regulation of SGLT1 activity. Metabolic labeling and immunoprecipitation with anti-SGLT1 specific antibodies did not show any significant changes in phosphorylation of SGLT1 protein. Tunicamycin to inhibit glycosylation reduced SGLT1 activity comparable to that seen with L-NAME treatment. The mechanism of inhibition was secondary to decreased affinity without a change in Vmax. Immunoblots of luminal membranes from tunicamycin treated or L-NAME treated IEC-18 cells showed a decrease in the apparent molecular size of SGLT1 protein to 62 and 67 kD, respectively suggesting an alteration in protein glycosylation. The deglycosylation assay with PNGase-F treatment reduced the apparent molecular size of the specific immunoreactive band of SGLT1 from control and L-NAME treated IEC-18 cells to approximately 62 kD from their original molecular size of 75 kD and 67 kD, respectively. Thus, the posttranslational mechanism responsible for the altered affinity of SGLT1 when cNO is diminished is secondary to altered glycosylation of SGLT1 protein. The intracellular pathway responsible for this alteration is cGMP and its dependent kinase.