The functionality of large-vessel endothelial cells, such as human umbilical vein endothelial cells (HUVEC), may differ significantly from that in the microvasculature. We established a method for the isolation of human colonic microvascular endothelial cells (HCMEC). Since colonic diseases are often accompanied by hypoxia we examined its effects on HCMEC of five individuals in comparison with HUVEC, with respect to the secretion of the soluble form of the two important vascular endothelial growth factor (VEGF) receptors, VEGFR-1 and 2. After dissociation by dispase/collagenase of mucosal and submucosal tissue obtained from normal adult colon, HCMEC were isolated using CD31-coated magnetic beads and cultivated as monolayers. Subsequent characterization studies demonstrated the endothelial phenotype, including VEGFR-1 and 2 mRNA and protein expression. sVEGFR expression analyses were performed using ELISA. Under hypoxic conditions significantly enhanced levels of sVEGFR-1 on HUVEC were observed (p<0.001), while in HCMEC there was a markedly variable reaction to hypoxia, with cases of enhanced, unchanged and reduced expression. sVEGFR-2 was significantly decreased in HCMEC under hypoxia (p<0.001). In contrast, the responses of sVEGFR-2 levels to hypoxia in HUVEC were variable, that is, either unchanged or up-regulated. The different secretion profiles of sVEGFR-1 and 2 between HUVEC and HCMEC under normoxia and hypoxia underline the importance of using a functionally adequate and relevant microvasculature for in vitro studies of colonic diseases. The homogeneously reduced sVEGFR-2 levels in hypoxic HCMEC provide evidence for a novel microvascular endothelium-specific biomarker in hypoxia-response processes.