Malaria infection starts when sporozoites are transmitted to the mammalian host during a mosquito bite. Sporozoites enter the blood circulation, reach the liver, and infect hepatocytes. The formation of a parasitophorous vacuole (PV) establishes their intracellular niche. Recently, two members of the 6-Cys domain protein family, P52 and P36, were each shown to play an important albeit nonessential role in Plasmodium berghei sporozoite infectivity for the rodent host. Here, we generated p52/p36-deficient Plasmodium yoelii parasites by the simultaneous deletion of both genes using a single genetic manipulation. p52/p36-deficient parasites exhibited normal progression through the life cycle during blood-stage infection, transmission to mosquitoes, mosquito-stage development, and sporozoite infection of the salivary glands. p52/p36-deficient sporozoites also showed normal motility and cell traversal activity. However, immunofluorescence analysis and electron microscopic observations revealed that p52/p36-deficient parasites did not form a PV within hepatocytes in vitro and in vivo. The p52/p36-deficient parasites localized as free entities in the host cell cytoplasm or the host cell nucleoplasm and did not develop as liver stages. Consequently, they did not cause blood-stage infections even at high sporozoite inoculation doses. Mice immunized with p52/p36-deficient sporozoites were completely protected against infectious sporozoite challenge. Our results demonstrate for the first time the generation of two-locus gene deletion-attenuated parasites that infect the liver but do not progress to blood-stage infection. The study will critically guide the design of Plasmodium falciparum live attenuated malaria vaccines.