Malaria parasites transmitted by mosquito bite are remarkably efficient in establishing human infections. The infection process requires roughly 30 minutes and is highly complex as quiescent sporozoites injected with mosquito saliva must be rapidly activated in the skin, migrate through the body, and infect the liver. This process is poorly understood for Plasmodium vivax due to low infectivity in the in vitro models. To study this skin-to-liver-stage of malaria, we used quantitative bioassays coupled with transcriptomics to evaluate parasite changes linked with mammalian microenvironmental factors. Our in vitro phenotyping and RNA-seq analyses revealed key microenvironmental relationships with distinct biological functions. Most notable, preservation of sporozoite quiescence by exposure to insect-like factors coupled with strategic activation limits untimely activation of invasion-associated genes to dramatically increase hepatocyte invasion rates. We also report the first transcriptomic analysis of the P. vivax sporozoite interaction in salivary glands identifying 118 infection-related differentially-regulated Anopheles dirus genes. These results provide important new insights in malaria parasite biology and identify priority targets for antimalarial therapeutic interventions to block P. vivax infection.