Spirochetes belonging to the Borrelia burgdorferi sensu lato (s. l.) complex have evolved remarkable ability to survive in diverse ecological niches during transmission cycles between ticks and vertebrate hosts by variable gene expression. To understand the events during spirochete transmission from feeding ticks to hosts, mRNA levels of selected B. afzelii genes (bbk32, dbpA, ospA, ospC and vlsE) were measured by quantitative real-time SYBR Green PCR. B. afzelii infected Ixodes ricinus nymphs fed on laboratory BALB/c mice for 0, 24, 48, and 72 hours. The mRNA levels of the constantly expressed flagellin gene were used for the relative quantification of selected genes. Differences in gene expression profiles were observed in unfed ticks and during tick feeding. mRNA levels of bbk32 and dbpA showed distinctive decreasing patterns during the first 24 hours post-attachment, while ospC and vlsE mRNA levels increased significantly during the feeding process. In contrast, ospA levels decreased for the 48 hours of tick feeding and slightly increased by 72 hours. More detailed and comprehensive studies on regulation of gene expression in different Borellia genospecies on the vector-host interface would aid to develop effective strategies in preventing pathogen transmission.