Protein-losing enteropathy (PLE), the excessive loss of plasma proteins through the intestine, often correlates with the episodic loss of heparan sulfate (HS) proteoglycans (HSPG) from the basolateral surface of intestinal epithelial cells. PLE onset is often associated with a pro-inflammatory state. We investigated whether loss of HS or treatment with the pro-inflammatory cytokine TNF directly causes protein leakage, and whether a combination of both exacerbates this process. We established the first in vitro model of PLE, and measured the flux of albumin:FITC through a monolayer of intestinal HT29 or Caco-2 cells grown on transwells and determined the integrity by transepithelial electrical resistance (TER). Loss of HS from the basolateral surface, either by heparanase digestion or by inhibition of HS synthesis, increased albumin flux 1.58±0.09-fold and reduced TER by 23.4±6.5%. TNF treatment increased albumin flux 4.04±0.03-fold and reduced TER by 75.7±4.7%, but only slightly decreased HS content. The combined effects of HS loss and TNF treatment were not only additive, but synergistic, with a 7.00±0.11-fold increase in albumin flux and a 83.9±8.1% reduction of TER. Co-incubation of TNF with soluble HS or heparin abolished these synergistic effects. Loss of basolateral HS directly causes protein leakage and amplifies the effects of the pro-inflammatory cytokine TNF. Our findings imply that loss of HSPGs renders patients more susceptible for PLE and offer a potential explanation for the favorable response some PLE patients have to heparin therapy.