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/m² 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.