In yeasts, the end-binding protein 1 (EB1) homologs regulate microtubule dynamics, cell polarization, and chromosome stability.However, the functions of EB1 orthologs in plant pathogenic fungi have not been characterized yet.Here, we observed that the FgEB1 deletion mutant (ΔFgEB1) of Fusarium graminearum exhibits twisted hyphae, increased hyphal branching and curved conidia, indicating that FgEB1 is involved in the regulation of cellular polarity.Microscopic examination further showed that the microtubules of ΔFgEB1 exhibited less organized in comparison with those of the wild type.In addition, the lack of FgEB1 also altered the distribution of polarity-related class I myosin via the interaction with the actin.On the other hand, we identified four core septins as FgEB1-interacting proteins and found that FgEB1-septin complex regulates conidial polar growth in the opposite orientation.Interestingly, FgEB1 and FgKar9 constitute another complex that modulates the response to carbendazim, a microtubule-damaging agent specifically.In addition, the deletion of FgEB1 led to dramatically decreased deoxynivalenol (DON) biosynthesis.Taken together, results of this study indicate that FgEB1 regulates cellular polarity, fungicide sensitivity and DON biosynthesis via different interactors in F.graminarum, which provides a novel insight into understanding of the biological functions of EB1 in filamentous fungi.