Hesperetin glucosides such as hesperidin and hesperetin-7-glucoside are abundantly present in citrus fruits and have various pharmacological properties. However, the potential toxicity of hesperetin glucosides remains unclear. An initial assessment of the safety of hesperetin-7-glucoside-β-cyclodextrin inclusion complex (HPTGCD) as a functional food ingredient was undertaken to assess toxicity and mutagenic potential. A bacterial reverse mutation assay (Ames test) using Salmonella typhimurium (strains TA98, TA1535, TA100, and TA1537) and Escherichia coli (strain WP2 uvrA) with HPTGCD (up to 5000 µg/plate) in the absence and presence of metabolic activation was negative. In a single oral (gavage) toxicity study in male and female rats, HPTGCD at dose up to 2000 mg/kg did not produce mortality nor clinical signs of toxicity or change in body weight. In a subchronic oral (dietary admix) toxicity study in rats receiving 0, 1.5, 3, and 5% HPTGCD for 13 weeks, no adverse effects were noted and the no-observed-adverse-effect level (NOAEL) was 5% in the diet (equivalent to 3267.7 mg/kg/day for males and to 3652.4 mg/kg/day for females). These results provide initial evidence of the safety of HPTGCD.