Airway inflammation leads to increased intracellular Ca²⁺ ([Ca²⁺]_i) levels in airway smooth muscle (ASM) cells. Sarcoplasmic reticulum (SR) Ca2+ release and reuptake are key components of ASM [Ca²⁺]_i regulation. Ca²⁺ reuptake occurs via sarcoendoplasmic reticulum Ca2+ ATPase (SERCA), and is regulated by the inhibitory protein phospholamban (PLB) in many cell types. In human ASM, we tested the hypothesis that inflammation increases PLB, thus inhibiting SERCA function, and leading to maintained [Ca²⁺]_i levels. Surprisingly, we found that human ASM does not express PLB protein (although mRNA is detectable). Overnight exposure to the pro-inflammatory cytokines tumor necrosis factor (TNF-) and interleukin-13 (IL-13) did not induce PLB expression, raising the issue of how SERCA is regulated. We then found that direct SERCA phosphorylation (via CaMKII) occurs in human ASM. In fura-2 loaded human ASM cells, we found that the CaMKII antagonist KN-93 significantly slowed the rate of fall of [Ca²⁺]_i transients induced by ACh or bradykinin (in zero extracellular Ca²⁺), suggesting a role for CaMKII-mediated SERCA regulation. SERCA expression was decreased by cytokine exposure, and the rate of fall of [Ca²⁺]_i transients was slowed in cells exposed to TNF- and IL-13. Cytokine effects on Ca²⁺ reuptake were unaffected by additional exposure to KN-93. These data indicate that in human ASM, SERCA is regulated by mechanisms such as CaMKII, and that airway inflammation maintains [Ca²⁺]_i levels by decreasing SERCA expression, and slowing Ca²⁺ reuptake.