Our previous study demonstrated mechanical stretch (MS) could induce the apoptosis of retinal pigment epithelial (RPE) cells, but the related mechanisms remained unclear. This study was to characterize the protein expression profile in RPE cell line ARPE-19 exposed to MS, cytochalasin D (CD; an inhibitor of actin polymerization) or CD + MS at 2-time points (6, 24 hr; n = 3, at each time point) by using proteomics technique. Our data highlighted that compared with control, ECE1 was continuously downregulated in ARPE-19 cells treated by MS or CD + MS from 6 to 24 hr. Function and protein-protein interaction network analyses showed ATAD2 was downregulated in all three treatment groups compared with control, but successive upregulation of RPS13 and RPL7 and downregulation of AHSG were specifically induced by MS. ATAD2 was enriched in cell cycle; AHSG was associated with membrane organization; RPS13 and RPL7 participated in ribosome biogenesis. Furthermore, transcription factor CREB1 that was upregulated in MS group at 24 hr after treatment, may negatively regulate ATAD2. The expressions of all crucial proteins in ARPE-19 cells were confirmed by western blot analysis. Overexpression of ATAD2 and AHSG were also shown to reverse the apoptosis of ARPE-19 cells induced by MS or CD + MS, with significantly decreased apoptotic rates and caspase-3 activities. Accordingly, our findings suggest downregulation of ATAD2 and AHSG may be potential contributors to the apoptosis of RPE cells induced by MS. Overexpression of them may represent underlying preventive and therapeutic strategies for MS-induced retinal disorders.