Calcium (Ca ) signaling modulates sodium (Na ) transport in plants; however, the role of the Ca sensor calmodulin (CaM) in salt tolerance is elusive. We previously identified a salt-responsive calmodulin (HvCaM1) in a proteome study of barley ( ) roots. Here, we employed bioinformatic, physiological, molecular, and biochemical approaches to determine the role of HvCaM1 in barley salt tolerance. CaM1s are highly conserved in green plants and probably originated from ancestors of green algae of the Chlamydomonadales order. was mainly expressed in roots and was significantly up-regulated in response to long-term salt stress. Localization analyses revealed that HvCaM1 is an intracellular signaling protein that localizes to the root stele and vascular systems of barley. After treatment with 200 mm NaCl for 4 weeks, knockdown (RNA interference) lines showed significantly larger biomass but lower Na concentration, xylem Na loading, and Na transportation rates in shoots compared with overexpression lines and wild-type plants. Thus, we propose that is involved in regulating Na transport, probably via certain class I high-affinity potassium transporter (HvHKT1;5 and HvHKT1;1)-mediated Na translocation in roots. Moreover, we demonstrated that HvCaM1 interacted with a CaM-binding transcription activator (HvCAMTA4), which may be a critical factor in the regulation of in barley. We conclude that HvCaM1 negatively regulates salt tolerance, probably via interaction with HvCAMTA4 to modulate the down-regulation of and/or the up-regulation of to reduce shoot Na accumulation under salt stress in barley.