Abstract Background Cell replacement therapies have been proposed as possible alternatives to the current treatments for controlling blood glucose in insulin-dependent diabetes. Beta cells, however, often lose their glucose-stimulated insulin secretion (GSIS) when maintained for prolonged periods in culture. For beta cell lines to be considered as a suitable source of transplantable tissue, it is essential that their GSIS is maintained. This study aimed to investigate cellular events involved in this loss of GSIS, to enable future optimization and enhancement of this response. Methods GSIS was investigated in low and high-passage murine insulinoma MIN-6 cells (using in vitro static procedures) and assessing levels of secreted (pro)insulin by enzyme-linked immunosorbent assays. Expression of relevant islet gene transcripts, including insulin, glucagon, somatostatin, and pancreatic polypeptide, was investigated by RT-PCR analysis. Results At low-passage, MIN-6 cells produced an approximately four- to fivefold increase in (pro)insulin secretion in response to 26.7 mmol/L glucose compared to 3.3 mmol/L glucose; at high passage, this response was lost. Expression of glucagon and somatostatin mRNAs were down-regulated with increased passage, while levels of insulin and pancreatic polypeptide mRNAs were apparently unchanged. Conclusion The maintenance of insulin mRNA levels in high-passage MIN-6 cells with down-regulation of glucagon (stimulates insulin secretion) and somatostatin (inhibits insulin secretion) gene transcript levels suggests that these cells have not lost their ability to maintain insulin production, but that the loss of glucose responsiveness may be due to a general effect on regulated secretion. Further studies investigating the regulated secretory pathway in these cells may further explain the mechanistic changes occurring with passaging of beta cells.