Dietary essential amino acids have an important influence on the lifespan and fitness of animals. The expression of the NAD(+)-dependent histone deacetylase, Sir2, can be influenced by diet, but its role in the extension of lifespan has recently been challenged. Here, we used the honeybee to test how the dietary balance of carbohydrates and essential amino acids and/or Sir2 affected lifespan. Using liquid diets varying in their ratio of essential amino acids to carbohydrate (EAA:C), we found that adult worker bees fed diets high in essential amino acids (≥1:10) had shorter lifespans than bees fed diets containing low levels of dietary amino acids. Bees fed a 1:500 EAA:C diet lived longer and, in contrast to bees fed any of the other diets, expressed Sir2 at levels tenfold higher or more than bees fed a 1:5 EAA:C diet. When bees were fed the 1:500 diet, small interfering RNA (siRNA)-mediated knock-down of Sir2 expression shortened lifespan but did not reduce survival to the same extent as the 1:5 diet, indicating that Sir2 contributes to mechanisms that determine lifespan in response to differences in macronutrient intake but is not the sole determinant. These data show that the ratio of dietary amino acids to carbohydrate influences Sir2 expression and clearly demonstrate that Sir2 is one of the factors that can determine honeybee lifespan. We propose that effects of dietary amino acids and Sir2 on lifespan may depend on the simultaneous activation of multiple nutrient sensors that respond to relative levels of essential amino acids and carbohydrates.