Administration of β-sitosterol (42 mg/kg per day) for 3 weeks to 8-month-old male LXRβ^−/− mice resulted in the death of motor neurons in the lumbar region of the spinal cord and loss of tyrosine hydroxylase-positive dopaminergic neurons in the substantia nigra. In mice at 5 months of age, β-sitosterol had no observed toxicity but at 16 months of age, it caused severe paralysis and symptoms typical of dopaminergic dysfunction in LXRβ^−/− mice. WT mice were not affected by these doses of β-sitosterol. In 5-month-old mice, levels of the intestinal transporters, ABCG5/8 and Niemann-Pick C1 Like 1, were not affected by loss of liver X receptor (LXR) β and/or treatment with β-sitosterol nor were there changes in plasma levels of cholesterol or β-sitosterol. In 8-month-old LXRβ^−/− mice there was activation of microglia in the substantia nigra pars reticulata and aggregates of ubiquitin and TDP-43 in the cytoplasm of large motor neurons in the lumbar spinal cord. Brain cholesterol concentrations were higher in LXRβ^−/− than in their WT counterparts, and treatment with β-sitosterol reduced brain cholesterol in both WT and LXRβ^−/− mice. In LXRβ^−/− mice but not in WT mice levels of 24-hydrocholesterol were increased upon β-sitosterol treatment. These data indicate that multiple mechanisms are involved in the sensitivity of LXRβ^−/− mice to β-sitosterol. These include activation of microglia, accumulation of protein aggregates in the cytoplasm of large motor neurons, and depletion of brain cholesterol.