The malaria parasite Plasmodium falciparum infects humans and first targets the liver where liver‐stage parasites undergo pre‐erythrocytic replication. Liver‐stage antigen‐1 (LSA‐1) is currently the only identified P. falciparum protein for which expression is restricted to liver stages. Yet, the importance of LSA‐1 for liver‐stage parasite development remains unknown. Here we deleted LSA‐1 in the NF54 strain of P. falciparum and analysed the lsa‐1^‐ parasites throughout their life cycle. lsa‐1^‐ sporozoites had normal gliding motility and invasion into hepatocytes. Six days after infection of a hepatocytic cell line, lsa‐1^‐ parasites exhibited a moderate phenotype with an ∼50% reduction of late liver‐stage forms when compared with wild type. Strikingly, lsa‐1^‐ parasites growing in SCID/Alb‐uPA mice with humanized livers showed a severe defect in late liver‐stage differentiation and exo‐erythrocytic merozoite formation 7 days after infection, a time point when wild‐type parasites develop into mature merozoites. The lsa‐1^‐ parasites also showed aberrant liver‐stage expression of key parasite proteins apical membrane antigen‐1 and circumsporozoite protein. Our data show that LSA‐1 plays a critical role during late liver‐stage schizogony and is thus important in the parasite transition from the liver to blood. LSA‐1 is the first P. falciparum protein identified to be required for this transitional stage of the parasite life cycle.