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Background: Rift Valley Fever virus (RVFV) is a zoonotic arbovirus of public health impact infecting livestock, wildlife, and humans mainly in Africa and other parts of the world. Despite its public health importance, mechanisms of RVFV maintenance during interepidemic periods (IEPS) remain unclear., Objective: We aimed to examine comparatively exposure to RVFV between humans and goats and RVFV infection between humans, goats and mosquitoes., Methods: A cross sectional study was performed in the Lower Moshi area of the Kilimanjaro region from March to June 2020. RVFV exposure was determined by detecting IgG/IgM to RVFV using a competitive enzyme linked immunosorbent assay whereas infection was determined by real time quantitative polymerase chain reaction (RT-qPCR) assay., Results: Results show that the male gender was related to RVFV seropositivity (χ 2  = 5.351; p=0.030). Being 50 years and above was related to seropositivity (χ 2  =14.430; p=0.006) whereas bed net use, larger numbers of persons living in the same house (>7 persons) and RVFV seropositivity in goats were related to higher seropositivity to RVFV among humans χ 2  =6.003; p=0.021, χ 2  =23.213; p < 0.001 and χ 2  =27.053; p < 0.001), respectively. By the use of RT-qPCR, goats exhibited the highest RVFV infection rate of 4.1%, followed by humans (2.6%), Ae. aegypti (2.3%), and Cx. pipiens complex(1.5%). Likewise, a higher proportion of goats (23.3%) were RVFV seropositive as compared with humans (13.2%)., Conclusion: Our findings suggest the Lower Moshi area as a potential hotspot for Rift Valley Fever (RVF), posing the danger of being a source of RVFV spread to other areas. Goats had the highest infection rate, suggesting goats as important hosts for virus maintenance during IEPs. We recommend the implementation of strategies that will warrant active RVF surveillance through the identification of RVF hotspots for targeted control of the disease.