We previously demonstrated that increase of procathepsin L secretion by human melanoma cells strongly increased their tumourigenicity and switched their phenotype from low to highly metastatic. Thus, we herein analysed whether it was possible to inhibit procathepsin L secretion using anti-cathepsin L ScFv. For this purpose, we produced different forms of fusion cathepsin L in prokaryotic or eukaryotic expression systems. An anti-cathepsin L monoclonal antibody (mAb), named 3D8, was isolated from mice immunized with purified procathepsin L-His. This 3D8 mAb interacted with an epitope localized on the 156-197 amino acid sequence of cathepsin L and recognized recombinant or native forms of cathepsin L synthesized by human melanoma cells. An active anti-cathepsin L ScFv was generated and characterized from 3D8 mAb heavy and light variable chains. Then, human melanoma cells were transiently co-transfected with 3D8 ScFv and cathepsin L cDNAs. Data demonstrated that increase of 3D8 ScFv expression in human melanoma cells totally inhibited procathepsin L secretion and induced accumulation of intracellular procathepsin L. Our results constitute the first demonstration that anti-cathepsin L ScFv could be used in human melanoma cells to inhibit procathepsin L secretion. This ScFv represents a new molecular tool to explore cell therapy of human melanomas.