Thymosinβ-4(Tβ4) is an actin-sequestering protein involved in tumor malignancy. Primary cilia, microtubule-based organelles, are present in most eukaryotic cells, which might be related to tumor cell transformation. Here, we investigated whether ciliogenesis is affected by Tβ4 in HeLa human cervical cancer cells. The inhibition of Tβ4 attenuated primary cilia formation. The frequency of cilia was increased by Tβ4 overexpression. When yeast two-hybrid assay was performed by using Tβ4 as a bait, we rescued nephronophthisis 3(NPHP3), one of the components of primary cilia. Interaction of Tβ4 with NPHP3 in mammalian cells was confirmed by GST-pulldown assay. Their intracellular co-localization was observed by immunofluorescence staining at peripheral surface of cells. In addition, the number of ciliated cells was reduced by the inhibition of NPHP3. Moreover, NPHP3 expression was decreased by the inhibition of Tβ4 but it was increased by Tβ4 overexpression. Taken together, the results demonstrate that primary cilia formation could be regulated by Tβ4 through its interaction with NPHP3 and/or the control of NPHP3 expression. It suggests that Tβ4 is a novel regulator for primary cilia formation by NPHP3. It also suggests that tumorigenesis could be associated with inappropriate regulation of Tβ4 and/or NPHP3 expression to maintain primary cilia formation normally.