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 shown to each play an important albeit non-essential role in Plasmodium berghei sporozoite infectivity for the rodent host. Here, we generated p52(-)/p36(-) deficient Plasmodium yoelii parasites by simultaneous deletion of both genes using a single genetic manipulation. p52(-)/p36(-) 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(-) sporozoites also showed normal motility and cell traversal activity. However, immunofluorescence analysis and electron microscopic observations revealed that p52(-)/p36(-) parasites did not form a PV within hepatocytes in vitro and in vivo. The p52(-)/p36(-) 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(-) sporozoites were completely protected against infectious sporozoite challenge. Our results demonstrate for the first time the generation of two-loci gene deletion-attenuated parasites that infect the liver but do not progress to blood stage infection. The study will critically guide the design of P. falciparum live attenuated malaria vaccines.