Timely cytokinesis/septation is essential for hyphal growth and conidiation in Aspergillus nidulans. Genetic analyses have identified that A. nidulans has components of the septum initiation network (SIN) pathway; one of these, SEPH, is a key player for early events during cytokinesis. However, little is known about how the SEPH kinase cascade is regulated by other components. Here, we demonstrate that the phosphoribosyl pyrophosphate synthetase family acts antagonistically against the SIN so that the downregulation of AnPRS family can bypass the requirements of the SIN for septum formation and conidiation. The transcription defect of the Anprs gene family accompanied with the reduction of AnPRS activity causes the formation of hyper-septation as well as the restoration of septation and conidiation in the absence of SEPH. Clearly, the timing and positioning of septation is related to AnPRS activity. Moreover, with the extensive yeast two-hybrid analysis and rescue combination experiments, it demonstrated that AnPRS members are able to form the heterodimers for functional interacting entities but they appear to contribute so unequally that Anprs1 mutant display relatively normal septation, but Anprs2 deletion is lethal. Thus, compared to in yeast, the AnPRS family may have a unique regulation mechanism during septation in filamentous fungi.