Changes in the actin cytoskeleton, especially the formation of cross-linked actin networks (CLANs) are thought to contribute to the increased intraocular pressure observed in primary open-angle and steroid-induced glaucoma. To better understand the effects of glucocorticoids, we employed a shotgun method to analyze global changes in the cytoskeleton and integrin signaling pathways following dexamethasone (DEX) treatment of human trabecular meshwork (HTM) cells. RNA and cell lysates were obtained from HTM cells incubated with or without DEX. Changes in protein expression were determined by mass spectrometry (MS) following differential centrifugation of cell lysates to enrich for low-abundance cytoskeletal and signaling proteins, proteolytic digestion, and a titanium dioxide column to enrich for phosphopeptides. Results were validated by Western blots. Changes in RNA levels were determined with gene arrays and RT-PCR. Overall, MS identified 318 cytoskeleton associated proteins. Five of these proteins (PDLIM1, FGFR1OP, leiomodin-1, ZO-2 and LRP16A) were only detected in DEX-treated cells by MS. However, only PDLIM1 showed a statistically significant increase at the RNA level. Other proteins with differences at both the RNA and protein levels included β3 integrin, caveolin-1, Borg2, raftlin1, PI-3 kinase regulatory subunit α, transgelin, and filamin B. By immunofluorescence microscopy filamin B and PDLIM1 showed enhanced expression in human trabecular meshwork cells, but only PDLIM1 demonstrated significant localization within CLANs. Finally, MS showed that some of the cytoskeleton proteins (Borg2, leiomodin-1, LRP16A, raftlin1 and CKAP4) contained phosphorylated residues. This study suggests that DEX affects the expression of cytoskeleton proteins at the transcriptional and translational level and shows that a combined genomic and proteomic approach can be used for rapid analysis of proteins in the TM. It also shows that DEX altered the expression of components (PDLIM1 and β3 integrins) involved in CLAN formation and provides new findings into the effects of glucocorticoids on the cytoskeleton.