© 2020 Elsevier Inc. Hyaluronan synthase 2 (HAS2) is an integral membrane protein composed of multi-membrane-spanning regions and a large intracellular loop (HAS2-loop). We previously examined the effect of phorbol 12-myristate 13-acetate (PMA) and/or 4-methylumbelliferone (4-MU) on the synthesis of hyaluronan (HA) in human skin fibroblasts and found that TPA and 4-MU have opposing effects on HA synthesis by phosphorylation and O-linked β-N-acetylglucosaminylation of HAS2, respectively. In this study, we constructed an expression vector for the HAS2-loop and analyzed its post-translational modification by PMA and 4-MU using mass spectrometry. We identified a phosphorylation site at the position corresponding to the Thr328 position of full-length HAS2, which was detected in the cells regardless of the presence of PMA or 4-MU. We next prepared T328A site-directed mutagenesis construct-transfected cells and investigated HA synthesis. The amount of HA was increased in cells expressing full-length HAS2 compared to in mock cells, whereas the amount of HA synthesized by cells transfected with the T328A site-directed mutagenesis construct was the same as that in mock cells. This phosphorylation site corresponded with the casein kinase 1 substrate motif. These results suggest that Thr328 phosphorylation is an essential factor for HA synthesis by HAS2 and the role of HAS2-loop may be useful in analyzing the regulation of HAS2 synthesis in physiological and pathological conditions.