Human gastric carcinoma BGC-823 cells underwent morphological differentiation and cell cycle arrest in vitro when treated with 5 mM hexamethylene bisacetamide (HMBA) for 48 h. To further understand the mechanism of HMBA-induced differentiation, proteomic methodologies were applied to screen and identify altered proteins involved in the commitment of BGC-823 cells to differentiate. Five distinct altered proteins were acquired by two-dimensional (2-D) PAGE and were consequently identified as ras-related protein rab-35 (Rab-35), splice truncated isoform of transmembrane protease, serine 3 (serine TADG-12), regulator of G-protein signaling 1 (RGS1), ret finger protein-like 1 (RFPL1) and F-actin capping protein alpha-3 subunit (GSG3) by analysis of mass spectrograph. Of the five proteins, serine TADG-12 down-regulated under the detectable level after HMBA treatment, Rab-35, RGS1 and RFPL1 sharply up-regulated within the HMBA-induced BGC-823 cells, and GSG3, appearing in both treated and untreated cells, remarkably increased within BGC-823 cells after HMB Astimulation. Our results implicate that the molecular mechanism of BGC-823 cell differentiation in response to HMBA may involved in complex processes including a signaling network linking vesicle transport, actin cytoskeleton remodeling except for morphology differentiation, cell cycle G1 arrest.