Parkinson’s disease (PD) is a neurodegenerative disease characterized by the loss of dopaminergic neurons in the substantia nigra (SN) pars compacta, leading to motor and cognitive impairments. The lack of a cure for this disease highlights the urgency in finding drugs that can modify its course. This emphasizes the importance of creating preclinical models that mimic major aspects of the disease. Rotenone, a natural pesticide that inhibits the mitochondrial complex I, has been shown to induce inflammation and selectively destroys nigrostriatal dopaminergic cells and thus presents itself as a great candidate to generate an induced model of PD. The present study aims to establish and characterize an intrastriatal rotenone mouse model for PD. Mice injected with 2 or 3.6 µg/µL rotenone showed an increased number of ipsilateral rotations after amphetamine stimulation compared to vehicle-injected animals 2 and 3 weeks after striatal lesion. Moreover, 2 µg/µL rotenone-injected animals performed more contralateral than ipsilateral slips in the beam walk test over a period of 4 weeks; and 3.6 µg/µL rotenone-injected animals did significantly more contralateral slips than vehicle-injected animals 3 and 4 weeks after the injection. Further, both rotenone concentrations decreased the tyrosine hydroxylase (TH) levels, while increasing the Glial fibrillary acidic protein (GFAP) and Ionized calcium binding adaptor molecule 1 (Iba1) immunoreactive area in the ipsilateral SN. This intrastriatal rotenone mouse model is therefore a very robust and reproducible model of PD as it consistently mimics motor impairments, selective loss of dopaminergic neurons and neuroinflammation in the nigrostriatal pathway.