Abstract Background Although basement membrane was traditionally considered an inert barrier that tumour cells had to cross before invasion into the surrounding stroma, recent studies suggest that basement membrane components are not only degraded during tumour progression, but also newly synthesised at the invasive front. Objective This study aimed at evaluating (1) the expression of basement membrane proteins in human oral carcinogenesis and (2) the role that epithelial–mesenchymal interactions play on it, by using an in vitro oral cancer progression model. Material and methods In vitro three-dimensional (3D) organotypic cultures of normal, early neoplastic and neoplastic human oral mucosa were developed by growing primary normal human oral keratinocytes, dysplastic human oral keratinocytes (DOK cell line), and neoplastic human oral keratinocytes (PE/CA-PJ15 cell line) on type I collagen biomatrices, with or without primary fibroblasts isolated from normal human oral mucosa. The cultured tissues were immunohistochemically assessed for the expression of the major basement membrane proteins laminin-332, type IV collagen, and fibronectin. Results Expression of laminin-332, type IV collagen, and fibronectin was gradually more pronounced in neoplastic models when compared to normal mucosa models, and, with the exception of laminin-332, it was further enhanced by presence of fibroblasts. Deposition of type IV collagen at the epithelium–biomatrix interface occurred only in presence of fibroblasts, as well as the extracellular matrix deposition of fibronectin. Conclusions These findings, obtained in a 3D in vitro model that closely mirrors the in vivo human oral cancer progression, show an enhanced basement membrane protein expression during human oral cancer progression that is dependent on the epithelial–mesenchymal environment, respectively the existence of fibroblasts.