Breast cancer is an important public health problem, and its progression may be related to the extracellular matrix (ECM), which acts as a structural scaffold and instruction source for neoplastic cells. Laminins are ECM proteins regulating tumor biology. The laminin-derived peptide C16 regulates different properties of tumor cells. Here we analyzed C16-induced differential gene expression in MDA-MB-231 breast cancer cells. MCF-10A normal-like breast cells served as control. Among different cancer-related genes, C16 induced overexpression of GPNMB. This gene encodes a transmembrane protein GPNMB (glycoprotein non-metastatic B), involved with malignant phenotype of breast cancer cells. Immunoblot validated microarray results. To correlate gene and protein expression with cellular function, we investigated whether C16 would regulate invasion in breast cancer cells. siRNA experiments strongly suggested that C16 and GPNMB cooperate to regulate invasion of highly aggressive MDA-MB-231 cancer cells. We addressed regulatory mechanisms involved in C16-mediated increase of GPNMB protein levels in MDA-MB-231 cells, and observed that C16 stimulates β1 integrin and Src phosphorylation. Furthermore, Src inhibition decreases peptide-induced GPNMB expression levels. To contextualize in vivo our results in vitro, we addressed GPNMB immunostaining in breast cancer human tissue microarrays. Quantitative immunohistochemistry showed that GPNMB is significantly more expressed in breast cancer compared to normal tissue. We concluded that laminin-derived peptide C16 regulates gene and protein expression of GPNMB in breast cancer cells. C16 and GPNMB may cooperate to regulate invasion of highly aggressive MDA-MB-231 cells, probably through Src signaling. GPNMB presented increased expression in breast cancer in vivo compared to normal breast tissue.