Larvae of Galleria mellonella are widely used to study the virulence of microbial pathogens and for assessing the potency of antimicrobial agents. This work examined the effect of nutritional deprivation on the ability of larvae to withstand infection in order to establish standardized conditions for the treatment of larvae for in vivo testing. Larvae deprived of food for seven days demonstrated an increased susceptibility to infection by the yeast Candida albicans. These larvae displayed a lower density of hemocytes compared with controls but hemocytes from starved and control larvae demonstrated the same ability to kill yeast cells. Hemolymph from starved larvae demonstrated reduced expression of a range of antimicrobial peptides (e.g., lipocalin) and immune proteins (e.g., apolipophorin and arylphorin). Deprivation of G. mellonella larvae of food leads to a reduction in the cellular and immune responses and an increased susceptibility to infection. Researchers utilizing these larvae should ensure adequate food is provided to larvae in order to allow valid comparisons to be made between results from different laboratories.