Objective ·To investigate the regulatory role of microglial transient receptor potential vanilloid 1 (TRPV1) in the pathological progression of apolipoprotein E4 (ApoE4)-associated Parkinson's disease (PD). Methods ·APOE3/Trpv1^flox/flox (E3/Trpv1^fl/fl), APOE4/Trpv1^flox/flox (E4/Trpv1^fl/fl), and microglia-specific Trpv1 knockout Cx3cr1^Cre (E4/Trpv1^MGKO) mice were bred and injected with human A53T α-synuclein (AAV-hα-syn) into the substantia nigra pars compacta (SNpc). AAV-GFP was injected into the SNpc of E3/Trpv1^fl/fl mice as a control. After 30 days, motor function, coordination, and muscle endurance were evaluated using the open field test, traction test, pole test, rotarod test, and tail suspension test, while spatial learning and memory were assessed using the Morris water maze test. The immunofluorescence technique was used. The survival of dopaminergic neurons and pathological α-syn deposition in the SNpc were detected using tyrosine hydroxylase (TH) and phosphorylated Ser129 α-synuclein [p-α-syn (Ser129)] antibodies. The phagocytosis of microglia in the midbrain was detected using ionized calcium-binding adapter molecule 1 (Iba1) co-staining with p-α-syn (Ser129), while the accumulation of lipid droplets in microglia, neurons, and astrocytes was observed by BODIPY 493/503 staining. Results ·In traction tests, E4/Trpv1^MGKO(AAV-hα-syn) mice showed no significant differences in grip strength compared to E4/Trpv1^fl/fl(AAV-hα-syn) group. However, these mice exhibited increased mean velocity and total distance in open field tests, significantly prolonged time to turn (t_turn) and time for lateral ambulation (t_LA) in pole tests, shortened latency to remain on the rotarod, and induced stronger dystonic postures during tail suspension, indicating aggravated motor dysfunction in ApoE4-associated PD mice. The Morris water maze demonstrated that E4/Trpv1^MGKO(AAV-hα-syn) mice exhibited significantly prolonged escape latency and a reduced proportion of travel distance in the target quadrant, indicating that TRPV1 deficiency in microglia significantly impaired spatial learning ability in ApoE4-associated PD mice. Immunofluorescence revealed that the loss of dopaminergic neurons in the SNpc was aggravated in E4/Trpv1^MGKO(AAV-hα-syn) mice, and the p-α-syn deposition in the SNpc was increased. The phagocytosis of microglia in the mesencephalon was enhanced, and the accumulation of lipid droplets was increased. However, the deficiency of TRPV1 in microglia did not aggravate the accumulation of lipid droplets in astrocytes and neurons. Conclusion ·TRPV1 deficiency in microglia accelerates the pathological progression of ApoE4-associated PD and disrupts lipid metabolic homeostasis in microglia.