OBJECTIVE To identify non-housekeeping genes definitively expressed in the human trabecular meshwork (TM). METHODS Microarray gene expression data on TM cultured cells from four studies were compared. Genes that were queried in at least three studies and assessed to be expressed in at least three studies were considered definitively expressed genes of the human TM. Housekeeping genes were removed from this set of genes. The non-housekeeping TM gene profile was analyzed for pathway enrichment and microRNA targeting, using bioinformatics tools. The results were compared with results of previous non-array based studies. RESULTS Nine hundred and sixty-two genes were identified as non-housekeeping TM expressed genes. Analysis of these by Kyoto Encyclopedia of Genes and Genomes led to identification of two enriched biologic pathways that achieved a highly significant Bonferroni p-value (p≤0.01): focal adhesion and extracellular matrix (ECM)-receptor interaction. Many of the genes were previously implicated in TM-related functions and the TM-associated disease glaucoma; however, some are novel. MicroRNAs known to be expressed in the trabecular meshwork were predicted to target some of the genes. Ten genes identified here, ALDH1A1 (aldehyde dehydrogenase 1 family, member A1), CDH11 (cadherin 11, type 2, OB-cadherin), CXCR7 (chemokine (C-X-C motif) receptor 7), CHI3L1 (chitinase 3-like 1), FGF2 (fibroblast growth factor 2), GNG11 (guanine nucleotide binding protein [G protein], gamma 11), IGFBP5 (insulin-like growth factor binding protein 5), PTPRM (protein tyrosine phosphatase, receptor type, M), RGS5 (regulator of G-protein signaling 5), and TUSC3 (tumor suppressor candidate 3), were also reported as TM expressed genes in three earlier non-microarray based studies. CONCLUSIONS A transcriptome consisting of 962 non-housekeeping genes definitively expressed in the human TM was identified. Multiple genes and microRNAs are proposed for further study for a better understanding of TM physiology.