Tsetse flies, the vectors of trypanosomiasis, represent a threat to public health and economy in sub-Saharan Africa. Despite these concerns, information on temporal and spatial dynamics of tsetse and trypanosomes remain limited and may be a reason that control strategies are less effective. The current study assessed the temporal variation of the relative abundance of tsetse fly species and trypanosome prevalence in relation to climate in the Maasai Steppe of Tanzania in 2014–2015. Tsetse flies were captured using odor-baited Epsilon traps deployed in ten sites selected through random subsampling of the major vegetation types in the area. Fly species were identified morphologically and trypanosome species classified using PCR. The climate dataset was acquired from the African Flood and Drought Monitor repository. Three species of tsetse flies were identified: G. swynnertoni (70.8%), G. m. morsitans (23.4%), and G.pallidipes (5.8%). All species showed monthly changes in abundance with most of the flies collected in July. The relative abundance of G. m. morsitans and G. swynnertoni was negatively correlated with maximum and minimum temperature, respectively. Three trypanosome species were recorded: T. vivax (82.1%), T. brucei (8.93%), and T. congolense (3.57%). The peak of trypanosome infections in the flies was found in October and was three months after the tsetse abundance peak; prevalence was negatively correlated with tsetse abundance. A strong positive relationship was found between trypanosome prevalence and temperature. In conclusion, we find that trypanosome prevalence is dependent on fly availability, and temperature drives both tsetse fly relative abundance and trypanosome prevalence.