Keratoconus (KC) is a multi-factorial corneal ectasia with unknown etiology affecting approximately 1:2000 people worldwide. Dysregulated gene expression, using RNA-Seq technology, have been reported in KC corneal tissue. However, the differential expression of genes, in KC corneal stromal cells have been widely ignored. We utilized mRNA-Seq to analyze gene expression in primary human corneal stromal cells derived from five non-Keratoconus healthy (HCF) and four Keratoconus (HKC) donors. Selected genes were further validated using real time PCR (RT-PCR). We have identified 423 differentially expressed genes with 187 down- and 236 up-regulated in KC-affected corneal stromal cells. Gene ontology analysis using WebGestalt indicates the enrichment of genes involved in cell migration, extracellular matrix, adherens junction, and MAPK signaling. Our protein-protein interaction network analysis identified several network seeds, such as EGFR, NEDD4, SNTA1, LGALS3BP, HSPB1, SDC2, MME, and HIF1A. Our work provides an otherwise unknown information on the transcriptional changes in HKCs, and reveals critical mechanisms of the cellular compartment. It also highlights the importance of human-based in vitro studies on a disease that currently lacks strong biomarkers and animal models.