Caco-2 cells are increasingly used to study the absorption of drugs and nutrients, including D-glucose, an important nutrient that mainly gets absorbed from the intestine by the sodium/glucose cotransporter 1 (SGLT1). However, disadvantages of Caco-2 cells for such studies have been reported, e.g., D-glucose cannot elicit translocation of the intracellular pool of SGLT1 to the apical membrane, the origin of the cells affects glucose uptake, and Caco-2 cells exhibit heterogeneity. This study aimed to characterize SGLT1-mediated glucose transport across Caco-2 cell monolayers. We found that at lower glucose concentrations (5 mM) SGLT1 contributes more to total glucose transport than at higher (10 mM) glucose concentrations, suggesting contributions by another transporter at higher glucose concentrations. This contrasts with the in vivo situation, where SGLT1 dominant glucose transporter at all glucose concentrations. We also tested whether known regulators like sugars or catecholamines can stimulate glucose transport across Caco-2 cell monolayers. Neither epinephrine nor 2-deoxy-D-glucose could stimulate glucose transport. Moreover, the epinephrine could not induce accumulation of cyclic adenosine monophosphate (cAMP) in Caco-2 cells, indicating the absence of a functional β2-adrenoceptor in Caco-2 cells, which could explain the lack of epinephrine effect on glucose transport. Also, Caco-2 cells may lack some kinases required for increased SGLT1 transport. Overall, SGLT1-mediated glucose transport and its regulation in Caco-2 cells differ from that in vivo, and caution is advised when extrapolating glucose transport results obtained with this model to the in vivo situation.