Tubulointerstitial fibrosis –a common end-stage feature of chronic kidney disease- is characterized by the presence of renal myofibroblasts and excessive accumulation of extracellular matrix proteins (ECM) in the renal tubular interstitium. The cytokine transforming growth factor beta 1 (TGF-β1) promotes myofibroblast activation and ECM proteins expression through intracellular Smads activation, having an important role in renal fibrosis. We have recently reported that the heterozygous disruption of the TGF-β1 receptor activin receptor-like kinase 1 (ALK1) leads to an increase in TGF-β induced renal fibrosis after ureteral obstruction. Thus, we analyzed the effect of ALK1 heterozygosity in TGF-β1 induced signaling and its consequent fibrotic response in mouse embryonic fibroblasts (MEFs). We have analyzed Smad signaling pathaways in ALK1 heterozygous MEFs (ALK1 þ /-) and their respective controls (ALK1þ/þ) in basal conditions and after TGF-β1 treatment by Western blot and immunofluorescence. Moreover, we have analyzed collagen I, fibronectin and connective tissue growth factor (CTGF) expression in basal conditions, after TGF-β1 stimulation and after ALK5 inhibition –with SB431542- and Smad3 inhibition –with SIS3-. TGF-β1 stimulation induced Smad2 and Smad3 phosphorylation and Smad2/3 translocation into the nucleus in ALK1 þ / þ and ALK1 þ /- MEFs, being this increase higher in ALK1 þ /- MEFs. Basal Smad2 and Smad3 phosphorylation was higher in ALK1 heterozygous fibroblasts. TGF-β1 stimulation did not induce Smad1 phosphorylation in ALK1þ/- MEFs and a very small Smad1 phosphorylation in ALK1þ/- MEFs. ALK1 heterozygous MEFs expressed more collagen I, fibronectin and CTGF than their respective controls in basal conditions. Stimulation with TGF-β1 lead to an increase in collagen I, fibronectin and CTGF, being the increase in fibronectin and CTGF higher in ALK1þ/- MEFs. Inhibition of ALK5 and Smad3 reversed the ALK1þ/- phenotype. Summariz- ing, ALK1 heterozygous disruption leads to an impairment of TGF-β1 signaling and increased TGF-β1-induced fibrotic response in mouse embryonic fibroblasts.