The betA gene from Escherichia coli encoding choline dehydrogenase was transferred into elite maize inbred DH4866 via Agrobacterium-mediated transformation. Among five transgenic maize lines investigated, four lines, lines 2, 3, 4 and 5, accumulated significantly higher levels of glycinebetaine than wild type plants (non-transformants). At 10 and 15 °C, transgenic lines 2, 3, 4 and 5 exhibited higher germination capacity as determined by the germination speed and final germination percentage, and displayed less inhibition in seedling shoot growth rate than wild type plants. In chilling conditions, the chilling tolerance, expressed in cell membrane damage, degree of chilling injury, survival rate, and photosynthesis was enhanced in plants of transgenic lines 2, 3, 4 and 5 as compared with wild type plants. The increase of total soluble sugars and the increase of total free amino acids that was mostly due to accumulation of alanine, glutamic acid, glycine and serine might play an important role of osmoregulation under stress conditions.