As in plants sugar sensing and signal transduction involve pathways dependent or independent on hexokinase 1 (HXK1) as a glucose sensor, research was conducted to determine which pathway is responsible for regulation of the nitrate reduction. An Arabidopsis mutant with T-DNA insertion in the AtHXK1 gene and defects in glucose signaling (hxk1) was used to determine nitrate reductase (NR) activity, NIA genes expression in leaves after 8-h treatment with sugars (glucose and sucrose), organic acids [2-oxoglutarate (2OG)] and amino acids (glutamine and glutamate). Sugars, especially sucrose, caused induction of NR actual activity accompanied by an increase of the NR activation state, indicating the posttranslational nature of the modifications. Those modifications were observed in wild-type (WT) and hxk1 leaves, suggesting that regulation of NR activity by sugars does not involve HXK1 as a glucose sensor. Moreover, sugars enhanced expression of NIA genes. However, a higher level of NIA transcripts did not lead to an increase of total NR activity in sugar-treated plants. This may suggest that posttranslational modification of NR is fundamental regulatory mechanisms controlling NR activity in response to C metabolites. Treatment of plants with 2-OG also modified NR through the posttranslational modifications. Elevation of actual NR activity and the enzyme activation state in WT and hxk1 leaves was observed. Amino acids caused a decrease of NIA gene expression and NR activities in WT and hxk1 leaves indicating that mutation in the hexokinase-dependent glucose signaling pathway did not interrupt the amino acid feedback regulation of NR.