Abstract During chronic liver injury, regeneration occurs through hepatic progenitor cells (HPCs), which can generate both hepatocytes and biliary cells. Understanding the regulation of HPCs may offer therapeutic opportunities to augment liver regeneration. HPCs are associated with an increase in laminins in the extracellular matrix, leading to speculation that cell-matrix interactions may regulate HPC behaviour. Laminins are heterotrimeric proteins composed of an α, β, and γ chain. There are five α chains, with different cell surface receptor binding properties. We aimed to describe the laminin α chains associated with the HPC response, and to define the effect of different laminin chains on HPCs in vitro. We found that the laminin α chains are differentially regulated during HPC activation. There were significant increases in α2 and α5 chains in two independent murine models of regeneration, with downregulation of the α3 chain. Using dual immunofluorescence, we showed that HPCs are most closely associated with the laminin α5 chain. To look at the functional effects of matrix components on cell behaviour, we have used a line of spontaneously immortalised HPCs (bmols). Laminin α5 promotes HPC adhesion, spreading, and migration, compared with other α chains. These effects are partly blocked by antibodies against β1 integrin. Small-interfering RNA knockdown of laminin α5 results in hepatocytic differentiation, as demonstrated by increased albumin synthesis. Laminin α5-containing matrix is deposited around HPCs during regeneration and this supports cell attachment, migration, and maintenance of an undifferentiated phenotype in vitro. Funding UK Medical Research Council.