The study examined whether royal jelly (RJ) can prevent obesity and ameliorate hyperglycemia in type 2 diabetes. This study utilized obese/diabetic KK-Ay mice. RJ (10 mg/kg) was administered by oral gavage. Body weight, plasma glucose and insulin levels were measured. mRNA and protein levels were determined using quantitative reverse transcription polymerase chain reaction and western blotting, respectively. Four weeks of RJ administration improved hyperglycemia and partially suppressed body weight gain, although the latter effect did not reach statistical significance. In addition, RJ administration did not improve insulin resistance. RJ administration suppressed the mRNA expression of glucose-6-phosphatase (G6Pase), a key enzyme of gluconeogenesis, in the liver. Simultaneously, RJ administration induced adiponectin (AdipoQ) expression in abdominal fat, adiponectin receptor-1 (AdipoR1) expression in the liver and phosphorylated AMP-activated protein kinase (pAMPK) expression, which suppressed G6Pase levels in the livers of KK-Ay mice. pAMPK levels were also increased in skeletal muscle, but glucose transporter-4 (Glut4) translocation was not increased in the RJ supplementation group. The improvement in hyperglycemia due to long-term RJ administration may be because of the suppression of G6Pase expression through the upregulation of AdipoQ and AdipoR1 mRNA and pAMPK protein expressions.