Regulation of intracellular Ca²⁺ ([Ca²⁺]_i) in airway smooth muscle (ASM) involves sarcoplasmic reticulum (SR) Ca²⁺ release and reuptake via sarco-endoplasmic reticulum Ca²⁺ ATPase (SERCA). We examined regulation of SERCA in porcine ASM, hypothesizing that the regulatory protein phospholamban (PLN) and calmodulin (CaM)-CaM kinase (CaMKII) pathway play a role. In porcine ASM microsomes, we examined expression and extent of PLN phosphorylation following pharmacological inhibition of CaM (W7) vs. CaMKII (KN62/KN93), and found that PLN is phosphorylated by CaMKII. Using enzymatically-dissociated single ASM cells loaded with the Ca²⁺ indicator fluo-3 and imaged using fluorescence microscopy, the effects of PLN siRNA, W7 and KN62 on [Ca²⁺]_i responses to ACh and direct SR stimulation were measured. PLN siRNA slowed the rate of fall of [Ca²⁺]_i transients, as did both W7 and KN62. The two inhibitors additionally slowed reuptake in the absence of PLN. Pre-exposure to W7 or KN62 did not prevent initiation of ACh-induced [Ca²⁺]_i oscillations (previously shown to represent repetitive SR Ca²⁺ release/reuptake). However, once ACh-induced [Ca²⁺]_i oscillations reached steady-state, subsequent exposure to W7 or KN62 decreased oscillation frequency and amplitude and slowed the fall time of [Ca²⁺]_i transients, suggesting SERCA inhibition. Exposure to W7 completely abolished ongoing ACh-induced [Ca²⁺]_i oscillations. Pre-exposure to W7 or KN62 did not affect SR Ca²⁺ release induced by caffeine, indicating that RyR channels were not directly inhibited. These data indicate that in porcine ASM, the CaM-CaMKII pathway regulates SR Ca²⁺ reuptake, potentially through altered PLN phosphorylation.