To document the expansion of human babesiosis in Connecticut, we analyzed reservoir host sera for seroreactivity to Babesia microti Franca and reviewed Connecticut human surveillance case data collected during 2001–2010. Sera from white-footed mice, Peromyscus leucopus Rafinesque, from 10 towns in 5 counties, collected at 4–7-yr periods between 2001 and 2010, were tested for total immunoglobulins. The prevalence of B. microti-positive mice was compared with confirmed and probable human case reports tabulated by the Connecticut Department of Public Health. The highest babesiosis and rodent seroprevalence rates were in New London County, where this protozoan disease was first documented in the state. However, human cases and reservoir host infection increased significantly from 2001–2005 to 2005–2010 and in other parts of the state. Clinicians should be aware that the disease is not confined to long-established endemic areas of the state.