Renin-producing juxtaglomerular cells are connected to each other and to endothelial cells of afferent arterioles by gap junctions containing Connexin 40 (Cx40), abundantly expressed by these two cell types. Here, we generated mice with cell-specific deletion of Cx40 in endothelial and in renin-producing cells, as its global deletion caused local dissociation of renin-producing cells from endothelial cells, renin hypersecretion, and hypertension. In mice lacking endothelial Cx40, the blood pressure, renin-producing cell distribution, and the control of renin secretion were similar to wild-type mice. In contrast, mice deficient for Cx40 in renin-producing cells were hypertensive and these cells were ectopically localized. Although plasma renin activity and kidney renin mRNA levels of these mice were not different from controls, the negative regulation of renin secretion by pressure was inverted to a positive feedback in kidneys lacking Cx40 in renin-producing cells. Thus, our findings show that endothelial Cx40 is not essential for the control of renin expression and/or release. Cx40 in renin-producing cells is required for their correct positioning in the juxtaglomerular area and the control of renin secretion by pressure.