Host-cell invasion by Toxoplasma gondii is associated with the shedding of microneme-derived adhesins (MICs) from the parasite surface. MICs play diverse roles during host cell invasion and MICs shedding modulates these processes. The intramembrane shedding of MICs was described to be mediated by an anonymous proteolytic activity called microneme protein protease 1 (MPP1). In order to unravel the precise function of MPP1 activity and to define the responsible enzyme(s), we generated parasite lacking the candidate enzymes, rhomboid protein proteases 4 and 5 (ROM4 and/or ROM5). We demonstrate that ROM4 mediates the cleavage of MIC2, MIC6 and a proportion of AMA1, while ROM5 cleaves AMA1 family of proteins. ROM4 null parasites possessed enhanced attachment and were impaired in host-cell invasion; however, intracellular growth was not impaired. Moreover, the deletion of ROM5 revealed no major phenotypic consequence during in vitro growth. We also analyzed the essentiality of the AMA1 family of proteins by generating respective parasite mutants, and show that compensatory mechanisms exist in coordinating host-cell invasion process.