Aim In the brain, alterations in sphingolipid metabolism contribute to several neurologic disorders; however, their effect on astrocytes is largely unknown. Here, we identified bioactive sphingolipids that affect intracellular free calcium concentration ([Ca2+]i), mobility of peptidergic secretory vesicles, signaling pathways involved in alterations of calcium homeostasis and explored the relationship between the stimulus‐evoked increase in [Ca2+]i and attenuation of vesicle mobility. Methods Confocal time‐lapse images were acquired to explore [Ca2+]i signals, the mobility of fluorescently tagged peptidergic vesicles and the structural integrity of the microtubules and actin filaments before and after the addition of exogenous sphingolipids to astrocytes. Results Fingolimod (FTY720), a recently introduced therapeutic for multiple sclerosis, and sphingosine, a releasable constituent of membrane sphingolipids, evoked long‐lasting increases in [Ca2+]i in the presence and absence of extracellular Ca2+; the evoked responses were diminished in the absence of extracellular Ca2+. Activation of phospholipase C and inositol‐1,4,5‐triphosphate receptors was necessary and sufficient to evoke increases in [Ca2+]i as revealed by the pharmacologic inhibitors; Ca2+ flux from the extracellular space intensified these responses several fold. The lipid‐evoked increases in [Ca2+]i coincided with the attenuated vesicle mobility. High and positive correlation between increase in [Ca2+]i and decrease in peptidergic vesicle mobility was confirmed independently in astrocytes exposed to evoked, transient Ca2+ signaling triggered by purinergic and glutamatergic stimulation. Conclusion Exogenously added cell‐permeable sphingosine‐like lipids exert complex, Ca2+‐dependent effects on astrocytes and likely alter their homeostatic function in vivo. This article is protected by copyright. All rights reserved.