Functional interactions between the initial and collecting lymphatics, as well as the molecular players involved, remain elusive. In this study, we assessed the influence of nitric oxide (NO) on lymphatic fluid velocity and flow, using a mouse tail model that permits intravital microscopy and microlymphangiography. We found that NO synthase (NOS) inhibition decreased lymphatic fluid velocity in the initial lymphatics, without any effect on their morphology. Using the same model, we found a similar effect in eNOS-/- mice and in mice treated with a selective endothelial NOS (eNOS) inhibitor. Next, we uncoupled the superficial initial lymphatics from the deeper collecting lymphatics by ligating the latter and found that lymphatic fluid velocity in NOS-inhibited mice became equal to that in control animals. Surprisingly, lymphatic fluid velocity was significantly increased after ligating the collecting lymphatics, and there was a concomitant increase in injection rate and mean lymphatic vessel diameter. Our results provide the first in vivo evidence that eNOS affects function of the whole microlymphatic system and that it is regulated via the collecting lymphatics.