Abstract <p>Prostate cancer (PCa) is the second leading cause of cancer- related mortality in men, and its progression is often driven by dysregulated signaling pathways. The Hedgehog (Hh) signaling pathway plays a critical role in PCa development and progression. Higher cell proliferation in cancerous cells necessarily demands more nucleotide biosynthesis. Inosine Monophosphate Dehydrogenase (IMPDH) regulates the rate-limiting step in this process, and forms cytoophidia structures in cancer cell lines following inhibition by Ribavirin and/or Mycophenolic acid (MPA) treatments. In this study, we focused on the relationship between Hh signaling and IMPDH- based cytoophidia on the proliferation and migration of PC-3 cells. Our results indicated that cytoophidia structures (detected using Immunofluorescence technique) are associated with an active Hh signaling pathway (measured in terms of its activation indicator, GLI1 expression using western blot technique). Knockdown of ADP- ribosylation factor-like protein 13B (<i>ARL13B</i>), a key regulator of Hh signaling, found to be associated with reduced GLI1 expression, lower levels of the cytoophidia induced by ribavirin and a reduction in both cell proliferation and migration. However, knockdown of <i>ARL13B</i> has no effect on the expression of IMPDH. Regarding the results of this investigation, we propose for the first time that ARL13B might potentially regulate IMPDH-based cytoophidia formation with subsequent effects on cell proliferation and migration.</p>