Endothelial cells selectively release pro-coagulant and pro-inflammatory mediators stored in Weibel-Palade bodies (WPBs) in order to regulate vascular function1. WPBs provide an intracellular storage pool for the pro-thrombotic protein von Willebrand factor (vWF) and the pro-immunogenic protein P-selectin. Exposure to thrombotic or inflammatory stimuli induce WPBs exocytosis. However, inappropriate exocytosis of WPBs can promote the pro-thrombotic environment evident in cardiovascular disease. The mechanisms underlying differential cargo release in order to produce physiologically distinct responses are poorly understood. Recent studies have unravelled the role of some Rab GTPase family members in regulating the exocytosis of WPBs2. Here, we describe a novel Rab GTPase (Rab46:CRACR2A-a) in endothelial cells3 that has GTPase and Ca2+ binding activities and is located on WPBs. Super-resolution microscopy confirmed that individual WPBs contain vWF that is juxtaposed to Rab46 whilst quantitative imaging analysis suggested that Rab46 may regulate a subpopulation of WPBs because only 49% of WPBs were positive for both vWF and Rab46 (n/N=3/8). To explore the role of Rab46 in endothelial cells we used imaged-based analysis of differentially stimulated cells in the presence and absence of Rab46 expression. Our data indicated that WPBs are able to distinguish between histamine and thrombin signals. Interestingly, histamine (30µm) but not thrombin (2U/ml) stimulation evoked a movement of WPBs towards the MTOC. This trafficking was dependent of Rab46 as depletion of Rab46 with specifically targeted siRNAs reduced histamine-evoked WPB clustering (0.35 ± 0.05 siRNA treated vs 0.64 ± 0.04 control siRNA, p-value < 0.05). Furthermore, we demonstrated that retrograde WPB trafficking to the MTOC is dependent on the integrity of microtubules and dynein activity. Experiments performed with nocodazole, a microtubule disrupting agent and ciliobrevin, a dynein inhibitor, abolished histamine-dependent Rab46 clustering (0.9 ± 0.06 histamine vs 0.6 ± 0.06 nocodazole, vs 0.6 ± 0.01 ciliobrevin, p-value < 0.01). Values are means ± SEM, compared by ANOVA. To investigate the molecular mechanisms underlying Rab46-dependent retrograde trafficking we performed pull-down experiments of heterologous expressed Rab46 nucleotide-binding mutants and mass spectrometry analysis. Dynein heavy chain was identified as a candidate effector protein and further biochemical experiments confirmed the interaction between endogenous Rab46 and dynein both in presence or absence of histamine stimulation (n=3). Taken together, these results suggest that after acute histamine stimulation, dynein-bound Rab46 mediates retrograde transport of a subset of WPBs along microtubules to the MTOC. These observations indicate Rab46 as a key regulator of differential WPB cargo secretion and understanding the Rab46/WPB signalling axis could provide novel therapeutic targets for cardiovascular disease.