Cases of human Lyme borreliosis (LB) are registered in all administrative regions of Ukraine and in the Autonomous Republic of Crimea with an increasing tendency. The hard tick Ixodes ricinus was proved as the main vector of Borrelia burgdorferi s.l., the causative agents of LB. We identified active natural LB foci, in which almost 25% of the adult I. ricinus ticks contained borrelias and more than 30% of the human population were seropositive for Lyme borreliae. The role of Mus musculus, Microtus arvalis, Myodes glareolus, Apodemus agrarius, and A. sylvaticus in the transmission cycle of the LB agent(s) was revealed. Combined foci and cases of LB/TBE (tick-borne encephalitis) mixed infection were registered in 8 oblasts. A considerable spectrum of different clinical manifestations of LB has been found including erythema migrans in 64.9% of cases, neurologic symptoms in 26.1%, rheumatic affliction in 24.7%, and cardiovascular problems in 8.3%.