This dataverse contains the original data, quantification datasets, and custom code affiliated with Swaminathan et al., 2026: doi: https://doi.org/10.1101/2025.08.15.669933 Abstract: Ca2+ signaling and its regulation are important for endothelial cell (EC) function and signaling. Yet, the spatiotemporal organization of Ca2+ activity and its regulation across a vascular plexus is poorly understood in an in vivo mammalian context. To overcome this gap in knowledge, we developed an intravital imaging approach to resolve Ca2+ activity with single cell resolution in skin vasculature of adult mice via multiphoton microscopy. Here, we tracked thousands of Ca2+ events in the skin capillary plexus during homeostasis and observed signaling heterogeneity between ECs, with just over half displaying Ca2+ activity at any given time. Longitudinal tracking of the same mice revealed that the same capillary ECs maintain Ca2+ activity over days to weeks. Interestingly, activity dynamics, such as frequency and event duration, are not conserved at a single cell level but are maintained at an EC population level. Molecularly, conditional deletion of the gap junction protein Connexin 43 (Cx43cKO) in ECs lead to a subset of ECs displaying sustained Ca2+ activity, biasing signaling dynamics of the whole network towards chronically persistent activity over time. Sustained capillary Ca2+ activity resulted in vascular permeability and flow dysregulation. Lastly, through pharmacological targeting of known agonists/antagonists, we showed that inhibition of L-type Voltage Gated Ca2+ channels (VGCCs) non cell-autonomously restores Ca2+ activity, blood flow, and barrier function in Cx43cKO mice. Collectively, our work provides insight into the characteristics, extent, and regulation of Ca2+ activity in skin capillaries of live mice with unprecedented spatial and temporal resolution.