Ca2+ plays a pivotal role in nitric oxide (NO)-promoted stomatal closure. However, the function of Ca2+ in NO inhibition of blue light (BL)-induced stomatal opening remains largely unknown. Here, we analyzed the role of Ca2+ in the crosstalk between BL and NO signaling in Vicia faba L. guard cells. Extracellular Ca2+ modulated the BL-induced stomatal opening in a dose-dependent manner, and an application of 5 μM Ca2+ in the pipette solution significantly inhibited BL-activated K+ influx. Sodium nitroprusside (SNP), a NO donor, showed little effect on BL-induced K+ influx and stomatal opening response in the absence of extracellular Ca2+ , but K+ influx and stomatal opening were inhibited by SNP when Ca2+ was added to the bath solution. Interestingly, although both SNP and BL could activate the plasma membrane Ca2+ channels and induce the rise of cytosolic Ca2+ , the change in levels of Ca2+ channel activity and cytosolic Ca2+ concentration were different between SNP and BL treatments. SNP at 100 μM obviously activated the plasma membrane Ca2+ channels and induced cytosolic Ca2+ rise by 102.4%. In contrast, a BL pulse (100 μmol/m 2 per s for 30 s) slightly activated the Ca2+ channels and resulted in a Ca2+ rise of only 20.8%. Consistently, cytosolic Ca2+ promoted K+ influx at 0.5 μM or below, and significantly inhibited K+ influx at 5 μM or above. Taken together, our findings indicate that Ca2+ plays dual and distinctive roles in the crosstalk between BL and NO signaling in guard cells, mediating both the BL-induced K+ influx as an activator at a lower concentration and the NO-blocked K+ influx as an inhibitor at a higher concentration. Ca2 + plays a pivotal role in nitric oxide (NO) -promoted stomatal closure. However, the function of Ca2 + in NO inhibition of blue light (BL) -induced stomatal opening remains largely unknown. Here, we analyzed the role of Ca2 + in the crosstalk between BL and NO signaling in Vicia faba L. guard cells. Extracellular Ca2 + modulated the BL-induced stomatal opening in a dose-dependent manner, and an application of 5 μM Ca2 + in the pipette solution regulated inhibited BL-activated K + influx. Sodium nitroprusside (SNP), a NO donor, showed little effect on BL-induced K + influx and stomatal opening response in the absence of extracellular Ca2 +, but K + influx and stomatal opening were inhibited by SNP when Ca2 + was added to the bath solution. Interestingly, although both SNP and BL could activate the plasma membrane Ca2 + channels and induce the rise of cytosolic Ca2 +, the change in levels of Ca2 + channel activity and cytosolic Ca2 + concentration were different between SNP and BL treatments. SNP at 100 μM obviously activated the plasma membrane Ca2 + channels and induced cytosolic Ca2 + rise by 102.4%. In contrast, a BL pulse (100 μmol / m 2 pers for 30 s) slightly activated the Ca2 + channels and resulted in a Ca2 + rise of only 20.8%. Consistently, cytosolic Ca2 + promoted K + influx at 0.5 μM or below, and significantly inhibited K + influx at 5 μM or above. Taken together, our findings indicate that Ca2 + plays dual and distinctive roles in the crosstalk between BL and NO signaling in guard cells, mediating both the BL-induced K + influx as an activator at a lower concentration and the NO-blocked K + influx as an inhibitor at a higher concentration.