Neutrophils are the first line of host defense against fungal infection that sense microbe size and selectively release neutrophil extracellular traps (NETs) in response to hyphal Candida albicans but phagocytose small yeast. It is well known that NET formation depends on microtubule and actin networks. However, the molecular mechanisms by which dynamic cytoskeleton networks regulate NET formation are still unclear. Here we showed that a guanine nucleotide exchange factor Lfc, which is crucial in coupling microtubule dynamics to RhoA GTPase activation, is critical for NET formation. Wild-type neutrophils incubated with phorbol 12-myristate 13-acetate (PMA) formed NETs in spread form. In contrast, the majority of Lfc-deficient neutrophils exhibited diffused nuclear phenotype associated with neutrophil elastase translocation and histone cirtrullination under the same treatment. Interestingly, ROS production was comparable between PMA-incubated wild-type and Lfc-deficient neutrophils further indicating there were profound defects in the late stage of spread NET formation in Lfc-deficient neutrophils. Moreover, Lfc-deficient neutrophils were less able to control Candida albicans infection due to their incapability in spread NET formation. Overall, our results suggested Lfc is a critical regulator for spread NET formation in response to PMA or Candida albicans incubation.