Nitrogen is an essential soil nutrient for plants, and lack of nitrogen commonly limits plant growth. Soil nitrogen is typically available to plants in two inorganic forms: nitrate and ammonium. To better understand how nitrate and ammonium differentially affect plant metabolism and development, we performed transcriptional profiling of the shoots of ammonium-supplied and nitrate-supplied Arabidopsis thaliana plants. Seven genes encoding class III glutaredoxins were found to be strongly and specifically induced by nitrate. RNA silencing of four of these glutaredoxin genes (AtGRXS3/4/5/8) resulted in plants with increased primary root length (~25% longer than wild-type) and decreased sensitivity to nitrate-mediated inhibition of primary root growth. Increased primary root growth is also a well-characterized phenotype of many cytokinin-deficient plant lines. We determined that nitrate induction of glutaredoxin gene expression was dependent upon cytokinin signaling and that cytokinins could activate glutaredoxin gene expression independent of plant nitrate status. In addition, crosses between "long-root" cytokinin-deficient plants and "long-root" glutaredoxin-silenced plants generated hybrids that displayed no further increase in primary root length (i.e. epistasis). Collectively, these findings suggest that AtGRXS3/4/5/8 operate downstream of cytokinins in a signal transduction pathway that negatively regulates plant primary root growth in response to nitrate. This pathway could allow Arabidopsis to actively discriminate between different nitrogen sources in the soil, with the preferred nitrogen source, nitrate, acting to suppress primary root growth (vertical dimension) in concert with its well-characterized stimulatory effect on lateral root growth (horizontal dimension).