Vascular endothelial growth factor-C (VEGF-C) plays an important role in lymphangiogenesis, however functional responses of lymphatic vessels to VEGF-C have not been characterized. We tested the hypothesis that VEGF-C-induced activation of VEGFR-3 increases lymphatic pump output. We examined the in vivo pump activity of rat mesenteric collecting lymphatics using intravital microscopy, during basal conditions and treatment with 1 nM recombinant VEGF-C, the selective VEGFR-3 agonist, VEGF-C156S (1 nM), or 0.1 nM VEGF-A. Their specific responses were also analyzed during selective inhibition of VEGFR-3 with MAZ51. Contraction frequency, end diastolic diameter, end systolic diameter, stroke volume index, pump flow index, and ejection fraction were evaluated. We also assessed arteriolar diameter and microvascular extravasation of FITC-albumin. The results show that both VEGF-C and VEGF-C156S significantly increased contraction frequency, end diastolic diameter, stroke volume index, and pump flow index in a time-dependent manner. VEGF-A caused a different response characterized by a significantly increased stroke volume after 30 min. of treatment. MAZ51 (5µM) caused tonic constriction and decreased contraction frequency. In addition, 0.5 and 5 µM MAZ51 attenuated VEGF-C- and VEGF-C156S-induced lymphatic pump activation. VEGF-A caused vasodilation of arterioles, while VEGF-C and VEGF-C156S did not significantly alter arteriolar diameter. Also, VEGF-A and VEGF-C caused increased microvascular permeability, while VEGF-C156S did not. Our results demonstrate that VEGF-C increases lymphatic pumping through VEGFR-3. Furthermore, changes in microvascular hemodynamics are not required for VEGFR-3-mediated changes in lymphatic pump activity.