Na,K-ATPase and inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) can form a signaling microdomain that in the presence of ouabain triggers highly regular calcium oscillations. Downstream effects include NF-kappaB activation. Here we report that ankyrin B (Ank-B), expressed in most mammalian cells, plays a pivotal role in the function of the Na,K-ATPase/IP3R signaling microdomain. In studies performed on a monkey kidney cell line, we show that Ank-B co-precipitates with both Na,K-ATPase and IP3R. We identify the N terminus tail of the Na,K-ATPase catalytic subunit and the N-terminal portion 1-604 of the IP3R as novel binding sites for Ank-B. Knockdown of Ank-B with small interfering RNA reduced the expression of Ank-B to 15-30%. This down-regulation of Ank-B attenuated the interaction between Na,K-ATPase and IP3R, reduced the number of cells responding to pm doses of ouabain with calcium oscillations, altered the calcium oscillatory pattern, and abolished the ouabain effect on NF-kappaB. In contrast, Ank-B down-regulation had no effect on the ion transporting function of Na,K-ATPase and no effect on the distribution and apparent mobility of Na,K-ATPase in the plasma membrane.