The nigrostriatal system composed of the dorsal striatum and the substantia nigra (SN) is highly involved in the control of motor behavior. Various extremal and pathological conditions as well as social isolation may cause an impairment of locomotor function; however, corresponding alterations in the nigrostriatal dopaminergic pathway are far from full understanding. Here we analyzed the effect of 3-day hindlimb unloading (HU) and social isolation (SI) on the key players of dopamine transmission in the nigrostriatal system of CD1 mice. Three groups of mice were analyzed: group-housed (GH), SI, and HU. Our data showed a significant decrease in the expression of tyrosine hydroxylase (TH) in the SN and dorsal striatum of HU mice, but only in comparison with SI group that suggested attenuation of dopamine synthesis in response to HU, while TH phosphorylation was reduced in comparison with both GH and SI animals. SI also led to a decrease in TH phosphorylation in the dorsal striatum that pointed on an impact of isolation too. Expression of dopamine receptors D1 in the dorsal striatum of HU mice was increased suggesting a compensatory response, but the activity of downstream signaling pathways involving PKA and CREB was inhibited. But in the dorsal striatum of SI mice, expression of DA receptors and activity of downstream signaling was not affected. Obtained data let us to conclude that combination of short-term HU and isolation impaired dopamine transmission in the nigrostriatal system.