Mammalian cells contain three closely related heterochromatin protein 1 (HP1) isoforms, HP1α, β and γ, which, by analogy to their unique counterpart in Schizosaccharomyces pombe, have been implicated in gene silencing, genome stability and chromosome segregation. However, the individual importance of each isoform during normal cell cycle and disease has remained an unresolved issue. Here, we reveal that HP1α shows a proliferation-dependent regulation, which neither HP1β nor γ display. During transient cell cycle exit, the HP1α mRNA and protein levels diminish. Transient depletion of HP1α, but not HP1β or γ, in tumoural and primary human cells leads to defects in chromosome segregation. Notably, analysis of an annotated collection of samples derived from carcinomas reveals an overexpression of HP1α mRNA and protein, which correlates with clinical data and disease outcome. Our results unveil a specific expression pattern for the HP1α isoform, suggesting a unique function related to cell division and tumour growth. The overexpression of HP1α constitutes a new example of a potential epigenetic contribution to tumourigenesis that is of clinical interest for cancer prognosis.