The enzyme hexokinase (HXK) is present in virtually all living organisms and catalyzes a key step in carbohydrate metabolism, the phosphorylation of glucose to glucose-6-phosphate. In addition, HXKs can mediate sugar sensing and signaling in plant cells thereby affecting growth promotion, plant development and senescence. The tobacco (Nicotiana tabacum) HXK gene family consists of at least 10 members. To dissect their individual roles, nine HXK genes were isolated and subjected to RNAi-mediated suppression. While the gene silencing of most NtHXK genes produced no phenotypes, silencing of NtHXK1 produced a distinct phenotype with strong growth defects, leaf chlorosis and stunted leaf development. The determination of soluble sugars and starch in leaves revealed that NtHXK1 suppression generated a starch-excess phenotype. In addition, it was shown that NtHXK1 is able to replace the sensing function of AtHXK1 in the Arabidopsis mutant gin2-1 (glucose insensitive; AtHXK1 null mutant).