O-GlcNAcylation is a nutrient-responsive glycosylation that plays a pivotal role in transcriptional regulation. Human RNA polymerase II (Pol II) is extensively modified by O-linked N-acetylglucosamine (O-GlcNAc) on its unique C-terminal domain (CTD), which consists of 52 heptad repeats. One approach to understanding the function of glycosylated Pol II is to determine the mechanism of dynamic O-GlcNAcylation on the CTD. Here, we discovered that the Pol II CTD can be extensively O-GlcNAcylated in vitro and in cells. Efficient glycosylation requires a minimum of 20 heptad repeats of the CTD and more than half of the N-terminal domain of O-GlcNAc transferase (OGT). Under conditions of saturated sugar donor, we monitored the attachment of more than 20 residues of O-GlcNAc to the full-length CTD. Surprisingly, glycosylation on the periodic CTD follows a distributive mechanism, resulting in highly heterogeneous glycoforms. Our data suggest that initial O-GlcNAcylation can take place either on the proximal or on the distal region of the CTD, and subsequent glycosylation occurs similarly over the entire CTD with nonuniform distributions. Moreover, removal of O-GlcNAc from glycosylated CTD is also distributive and is independent of O-GlcNAcylation level. Our results suggest that O-GlcNAc cycling enzymes can employ a similar mechanism to react with other protein substrates on multiple sites. Distributive O-GlcNAcylation on Pol II provides another regulatory mechanism of transcription in response to fluctuating cellular conditions.