Malaria infection is initiated when Anopheline mosquitoes inject sporozoites into the skin. Further development requires that sporozoites reach the liver, invade hepatocytes and develop into exoerythrocytic forms. Sporozoites contact and traverse many cell types as they migrate from skin to liver, however, the mechanism by which they switch from a migratory mode to an invasive mode is unknown. Using a rodent model system, we show that sporozoites use the sulfation level of host heparan-sulfate proteoglycans (HSPGs) to navigate in the mammalian host. Sporozoites migrate through cells expressing low-sulfated HSPGs, such as those in skin and endothelium, while highly-sulfated HSPGs of hepatocytes activate sporozoites for invasion. A calcium-dependent protein kinase is critical for the switch to an invasive phenotype and the process is accompanied by proteolytic cleavage of the sporozoite’s major surface protein. These findings explain how sporozoites retain their infectivity for an organ that is far from their site of entry.