In several cell types, a regulated efflux of NAD⁺ across Connexin 43 hemichannels (Cx43 HC) can occur, and extracellular NAD⁺ (NAD⁺_e) affects cell-specific functions. We studied the capability of bone marrow-derived human mesenchymal stem cells (MSC) to release intracellular NAD⁺ through Cx43 HC. NAD⁺ efflux, quantified by a sensitive enzymatic cycling assay, was significantly upregulated by low extracellular Ca²⁺ (5–6-fold), by shear stress (13-fold), and by inflammatory conditions (3.1- and 2.5-fold in cells incubated with lipopolysaccharide (LPS) or at 39°C, respectively), as compared with untreated cells, whereas it was downregulated in Cx43-siRNA-transfected MSC (by 53%) and by cell-to-cell contact (by 45%). Further, we show that NAD⁺_e activates the purinergic receptor P2Y₁₁ and a cyclic adenosin monophosphate (cAMP)/cyclic ADP-ribose/[Ca²⁺]_i signaling cascade, involving the opening, unique to MSC, of L-type Ca²⁺ channels. Extracellular NAD⁺ enhanced nuclear translocation of cAMP/Ca²⁺-dependent transcription factors. Moreover, NAD⁺, either extracellularly added or autocrinally released, resulted in stimulation of MSC functions, including proliferation, migration, release of prostaglandin E₂ and cytokines, and downregulation of T lymphocyte proliferation compared with controls. No detectable modifications of MSC markers and of adipocyte or osteocyte differentiation were induced by NAD⁺_e. Controls included Cx43-siRNA transfected and/or NAD⁺-glycohydrolase-treated MSC (autocrine effects), and NAD⁺-untreated or P2Y₁₁-siRNA-transfected MSC (exogenous NAD⁺). These findings suggest a potential beneficial role of NAD⁺_e in modulating MSC functions relevant to MSC-based cell therapies.