Tight junctions are formed from a complex of different individual proteins. These complexes are expressed by epithelial cells and form an intercellular barrier which restricts and regulates paracellular permeability. Tight junction proteins have also been shown to be expressed in non-epithelial cells which do not form tight junctions, including astrocytes. The function(s) of these proteins within non-epithelial cells, however, remains unclear. This study aims to characterise the expression of tight junction proteins in astrocytes and investigate the function(s) of these proteins in these cells. The expression of the tight junction proteins occludin, claudin-5 and zonula occludens-1 (ZO-1) was characterised in vitro in both human primary astrocytes and the 1321N1 human astrocytoma cell line and in vivo in human autopsy brain samples. The function(s) of occludin was investigated using a pull-down protein binding assay and mass spectrometry analysis to identify putative binding partners for this protein in astrocytes. The current study demonstrates astrocytic and nuclear expression of occludin and ZO-1 in vitro and in vivo. The expression of claudin 5 in astrocytes remains difficult to determine due to contradictory evidence in which the astrocytic expression of this protein in vitro is not supported in vivo. Putative binding partners were also identified for the N- and C-terminal domains of occludin. Many of these proteins have functions in RNA metabolic processes, consequently their identification as putative occludin binding partners implicates occludin in functions beyond the formation of the tight junction complex. Although these interactions have not yet been validated, this study’s findings provide a platform upon which future research can be constructed.