Maltosyltrehalose syrup (TG4 syrup) is an enzymatically derived starch hydrolysate consisting of only glucose. The main components are maltotetraose (G4) and maltosyltrehalose (TG4). G4 is a common component of essentially all starch hydrolysates, and consists of only four glucose molecules with α-1,4 linkages. TG4 also consists of four glucose units, but the terminal glycosidic bond at the reducing end of the glucose chain is inverted, which results in the final bond being α-1,1. The two terminal glucose units form a trehalose disaccharide molecule, attached to a maltose molecule. The presence of the trehalose moiety in the TG4 molecule results in unique functional and technical properties from what is found in other α-1,4 linked oligosaccharides. This paper presents results of standardized toxicity studies of TG4 syrup, including an in vitro bacterial mutagenicity test, a 90-day oral feeding study in rats, and a 90-day oral toxicity (gavage) study in rats. Treatment with TG4 syrup resulted in no microbial mutagenic activity or growth inhibition in either Salmonella typhimurium or Escherichia coli, even at the maximum dose of 5,000 μg/plate. The NOAEL in the 90-day oral feeding study was calculated as 10% of TG4 syrup in the diet, which was equal to 6,818 and 7,464 mg/kg/day (dwb) in male and female rats, respectively. The 90-day oral gavage toxicity study had a NOAEL of 5,000 mg/kg/day (dwb) in male and female rats. Taken together these data showed that TG4 syrup was safe for use in these studies, suggesting it is safe for consumption by humans.