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Objective To study differential proteomics analysis of rat trabecular meshwork cells under mechanical stimulation. Methods The rat trabecular meshwork cells in logarithmic growth period were selected.Mechanical stretching was loaded by FX-5000 tension system. The total proteins were extracted and identified by shotgun-based mass spectrometry test and analysis. Differential expressed proteins between sample group and control group were analyzed by significance analysis of microarray (SAM) method. Gene Ontology (GO) annotation, GO enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were used to gain significantly enriched differential proteins.Results A total of 57 differential proteins were screened by SAM method, all of which were up-regulated and distributed in various regions of the cell, and were involved in the processes of cell development, immunomodulation, stimulus response, metabolism and cell adhesion. Twenty-four of them were significantlyenriched in 9 biological process GO terms and 2 KEGG pathways, mainly assigned to heat shock proteins and ribosome proteins. Conclusions The effect of mechanical strain on protein expression of rat trabecular meshwork cells was wide and complex. The enrichment analysis showed that heat shock proteins and ribosome proteins played an important role in the response protection and intraocular pressure regulation under mechanical strain.