Quantitative tracking of Borrelia afzelii has shown that its transmission cycle differs from the salivary route of B. burgdorferi transmission by Ixodes scapularis. Borrelia afzelii are abundant in the guts of unfed Ixodes ricinus nymphs and their numbers continuously decrease during feeding. In contrast, spirochetes are not present in the salivary glands. Borrelia afzelii transmission starts during the early stages of feeding, spirochetes could be detected in murine skin within 1 day of tick attachment. Tick saliva is not essential for B. afzelii infectivity, the main requirement for successful host colonization being a change in outer surface protein expression that occurs in the tick gut during feeding. Spirochetes in vertebrate mode are able to survive within the host even if the tick is not present. On the basis of our data we propose that a direct ‘gut-to-mouth’ route of infection appears to be the main route of B. afzelii transmission.ImportanceLyme borreliosis is the most common vector-borne disease in the USA and Europe. The disease is caused by the Borrelia spirochetes and is transmitted through Ixodes ticks. A better understanding of how Borrelia spirochetes are transmitted is crucial for development of efficient vaccines for preventing Lyme borreliosis. Here we present that the transmission of European B. afzelii spirochetes by I. ricinus ticks significantly differs from the model transmission cycle described for American B. burgdorferi/I. scapularis. We suggest that B. afzelii is not transmitted via salivary glands but most likely through the ‘midgut to mouthpart’ route. We further demonstrate that tick saliva is not important for B. afzelii transmission and infectivity. Therefore, we support early studies by Willy Burgdorfer, who proposed that Borrelia transmission occurs by regurgitation of infected gut contents. Our findings collectively point to the Borrelia-tick midgut interface as the correct target in our endeavours to combat Lyme borreliosis.