TGFbeta regulates cellular processes by binding to type I and type II TGFbeta receptors (TbetaRI and TbetaRII, respectively). In addition to these signaling receptors, endoglin is an accessory TGFbeta receptor that regulates TGFbeta signaling. Although there are two different alternatively spliced isoforms of endoglin, L-endoglin (L, long) and S-endoglin (S, short), little is known about the effects of S-endoglin isoform on TGFbeta signaling. Here, we have analyzed the TGFbeta1 signaling pathways and the effects of L- and S-endoglin in endoglin-deficient L6E9 cells. We found that TGFbeta activates two distinct TbetaRI-Smad signaling pathways: ALK1-Smad1-Id1 and ALK5-Smad2-PAI1, in these cells. Interestingly, L-endoglin enhanced the ALK1-Id1 pathway, while S-endoglin promoted the ALK5-PAI1 route. These effects on signaling are supported by biological effects on TGFbeta1-induced collagen I expression and inhibition of cell proliferation. Thus, while L-endoglin decreased TGFbeta1-induced collagen I and CTGF expression and increased TGFbeta1-induced proliferation, S-endoglin strongly increased TGFbeta1-induced collagen I and CTGF expression, and reduced TGFbeta1-induced cell proliferation.