Lymphatic vessels (LVs) are important in the regulation of tissue fluid homeostasis and the pathogenesis of tumor progression. We investigated the innervation of LVs and the response to agonists and antagonists of the autonomic nervous system in vivo. While skin-draining collecting LVs express muscarinic, α- and β-adrenergic receptors on lymphatic endothelial cells and smooth muscle cells, intestinal lacteals express only β-adrenergic receptors and muscarinic receptors on their smooth muscle cells. Quantitative in vivo near-infrared imaging of the exposed flank-collecting LV revealed that muscarinic and α-adrenergic agonists increased LV contractility, whereas activation of β-adrenergic receptors inhibited contractility and initiated nitric oxide (NO)-dependent vasodilation. Tumor-draining LVs were expanded and showed a higher innervation density and contractility that was reduced by treatment with atropine, phentolamine, and, most potently, isoproterenol. These findings likely have clinical implications given the impact of lymphatic fluid drainage on intratumoral fluid pressure and thus drug delivery.