The phosphorylation of H3.3 Serine 31 occurs during mitosis. In embryonic stem cells, H3.3 Serine 31 phosphorylation (H3.3S31ph) is enriched at both the pericentric regions and the telomeres. However, the function of H3.3S31ph in mammalian cells have been poorly studied. This in part is due to a lack of cell model to study mutations of H3.3. In this study, we have generated a model to specifically study mutations on H3.3 and have used this model to create H3.3S31 phospho-mutants. We have identified that the phosphomimic (H3.3S31E) mutant showed increased levels of heterochromatin markers such as H3K9me3 and ATRX at the telomeres. Despite the increase of heterochromatin levels, we also identified increased telomere transcripts. Together, our results show that H3.3S31ph is not only important for telomere heterochromatin maintenance, but also to regulate normal telomere function.