Megalin/LRP2 is a receptor that plays important roles in the physiology of several organs, such as kidney, lung, intestine, and gallbladder and also in the physiology of the nervous system. Megalin expression is reduced in diseases associated with fibrosis, including diabetic nephropathy, hepatic fibrosis and cholelithiasis, as well as in some breast and prostate cancers. One of the hallmarks of these conditions is the presence of the cytokine transforming growth factor beta (TGF-ss). Although TGF-ss has been implicated in the reduction of megalin levels, the molecular mechanism underlying this regulation is not well understood. Here, we show that treatment of two epithelial cell lines (from kidney and gallbladder) with TGF-ss1 is associated with decreased megalin mRNA and protein levels, and that these effects are reversed by inhibiting the TGF-ss1 type I receptor (TGF-ssRI). Based on in silico analyses, the two SMAD-binding elements (SBEs) in the megalin promoter are located at positions -57 and -605. Site-directed mutagenesis of the SBEs and chromatin immunoprecipitation (ChIP) experiments revealed that SMAD2/3 transcription factors interact with SBEs. Both the presence of SMAD2/3 and intact SBEs were associated with repression of the megalin promoter, in the absence as well in the presence of TGF-ss1. Also, reduced megalin expression and promoter activation triggered by high concentration of albumin are dependent on the expression of SMAD2/3. Interestingly, the histone deacetylase inhibitor Trichostatin A (TSA), which induces megalin expression, reduced the effects of TGF-ss1 on megalin mRNA levels. These data show the significance of TGF-ss and the SMAD2/3 signalling pathway in the regulation of megalin and explain the decreased megalin levels observed under conditions in which TGF-ss is upregulated, including fibrosis-associated diseases and cancer.