Understanding how plants sense and respond to changes in nitrogen availability is the first step toward developing strategies for biotechnological applications, such as improvement of nitrogen use efficiency. However, components involved in nitrogen signaling pathways remain poorly characterized. Calcium is a second messenger in signal transduction pathways in plants, and it has been indirectly implicated in nitrate responses. Using aequorin reporter plants, we show that nitrate treatments transiently increase cytoplasmic Ca²⁺ concentration. We found that nitrate also induces cytoplasmic concentration of inositol 1,4,5-trisphosphate. Increases in inositol 1,4,5-trisphosphate and cytoplasmic Ca²⁺ levels in response to nitrate treatments were blocked by U73122, a pharmacological inhibitor of phospholipase C, but not by the nonfunctional phospholipase C inhibitor analog U73343. In addition, increase in cytoplasmic Ca²⁺ levels in response to nitrate treatments was abolished in mutants of the nitrate transceptor NITRATE TRANSPORTER1.1/Arabidopsis (Arabidopsis thaliana) NITRATE TRANSPORTER1 PEPTIDE TRANSPORTER FAMILY6.3. Gene expression of nitrate-responsive genes was severely affected by pretreatments with Ca²⁺ channel blockers or phospholipase C inhibitors. These results indicate that Ca²⁺ acts as a second messenger in the nitrate signaling pathway of Arabidopsis. Our results suggest a model where NRT1.1/AtNPF6.3 and a phospholipase C activity mediate the increase of Ca²⁺ in response to nitrate required for changes in expression of prototypical nitrate-responsive genes.