Background Plasmodium sporozoites are the highly motile forms of malaria-causing parasites that are transmitted by the mosquito to the vertebrate host. Sporozoites need to enter and cross several cellular and tissue barriers for which they employ a set of surface proteins. Three of these proteins are members of the thrombospondin related anonymous protein (TRAP) family. Here, potential additive, synergistic or antagonistic roles of these adhesion proteins were investigated. Methods Four transgenic Plasmodium berghei parasite lines that lacked two or all three of the TRAP family adhesins TRAP, TLP and TREP were generated using positive–negative selection. The parasite lines were investigated for their capacity to attach to and move on glass, their ability to egress from oocysts and their capacity to enter mosquito salivary glands. One strain was in addition interrogated for its capacity to infect mice. Results The major phenotype of the TRAP single gene deletion dominates additional gene deletion phenotypes. All parasite lines including the one lacking all three proteins were able to conduct some form of active, if unproductive movement. Conclusions The individual TRAP-family adhesins appear to play functionally distinct roles during motility and infection. Other proteins must contribute to substrate adhesion and gliding motility. Graphical Abstract