Vascular endothelial growth factor (Vegf) plays central roles in the establishment of stereotypic vascular patterning in vertebrates. However, it is not fully understood how the network of blood vessels is established and maintained during vascular development. A zebrafish ko095 mutant presented the disorganized vessels with abnormal branching of the established intersegmental vessels (ISVs) after 60 hours postfertilization. The gene responsible for ko095 encodes seryl-tRNA synthetase (Sars) with a nonsense mutation. The abnormal branching of ISVs in ko095 mutant was suppressed by the introduction of either wild-type Sars or a mutant Sars (T429A) lacking the enzymatic activity that catalyzes aminoacylation of transfer RNA for serine (canonical activity), suggesting that the abnormal branching is attributable to the loss of function of Sars besides its canonical activity. We further found the increased expression of vegfa in ko095 mutant at 72 hours postfertilization, which was also reversed by the introduction of Sars (T429A). Furthermore, the abnormal branching of ISVs in the mutant was suppressed by knockdown of vegfa or vegfr2 (kdra and kdrb). Knockdown of vegfc or vegfr3 rescued the abnormal ISV branching in ko095 mutant. These results suggest that the abnormal ISV branching in ko095 mutant is caused by the activated Vegfa-Vegfr2 signal and requires the Vegfc-Vegfr3 signal, because the latter is needed for general angiogenesis. Hence, we conclude that noncanonical activity of Sars is involved in vascular development presumably by modulating the expression of vegfa.