Objective:To investigate the mechanism of regulation of diacylglycerol kinase (DGK) on Hedgehog signaling pathway. Methods: The interaction between DGKs and IFT88 was analyzed through co-immunoprecipitation. The expression levels of Hedgehog signaling target gene Gli1 were detected after siRNA knockdown of DGKs. Ciliogenesis of MEF cells with DGKδ defeciency was observed under confocal microscope. The localization of IFT88 in primary cilia was detected as well in those DGKδ knockout MEFs. Results: The 7 members of DGK family were bound with IFT88 specifically. SiRNA knockdown of DGK decreased the transcriptional level of Gli1. DGKs regulated Hedgehog pathway between Ptch1 and Smo. Knockout of DGKδ inhibited growth of primary cilia and the distribution of IFT88 in cilia. Conclusion: Knockdown of DGK inhibits the activity of the Hedgehog signaling pathway, which is related to defect of ciliogenesis.