Background: The Rift Valley fever virus (RVFV) is an arthropod-borne phlebovirus. RVFV mostly causes outbreaks among domestic ruminants with a major economic impact. Human infections are associated with these events, with a fatality rate of 0.5-2%. Since the virus is able to use many mosquito species of temperate climates as vectors, it has a high potential to spread to outside Africa. Methodology/Principal Findings: We conducted a stratified, cross-sectional sero-prevalence survey in 1228 participants from Mbeya region, southwestern Tanzania. Samples were selected from 17,872 persons who took part in a cohort study in 2007 and 2008. RVFV IgG status was determined by indirect immunofluorescence. Possible risk factors were analyzed using uni-and multi-variable Poisson regression models. We found a unique local maximum of RVFV IgG prevalence of 29.3% in a study site close to Lake Malawi (N = 150). The overall seroprevalence was 5.2%. Seropositivity was significantly associated with higher age, lower socio-economic status, ownership of cattle and decreased with distance to Lake Malawi. A high vegetation density, higher minimum and lower maximum temperatures were found to be associated with RVFV IgG positivity. Altitude of residence, especially on a small scale in the high-prevalence area was strongly correlated (PR 0.87 per meter, 95% CI = 0.80-0.94). Abundant surface water collections are present in the lower areas of the high-prevalence site. RVF has not been diagnosed clinically, nor an outbreak detected in the high-prevalence area. Conclusions: RVFV is probably circulating endemically in the region. The presence of cattle, dense vegetation and temperate conditions favour mosquito propagation and virus replication in the vector and seem to play major roles in virus transmission and circulation. The environmental risk-factors that we identified could serve to more exactly determine areas at risk for RVFV endemicity.