Toll-like receptor 4 (TLR4) activation contributes to vascular dysfunction in pathological conditions such as hypertension and diabetes, but the role of chronic TLR4 activation on renal autoregulatory behavior is unknown. We hypothesized that subclinical TLR4 stimulation with low-dose lipopolysaccharide (LPS) infusion increases TLR4 activation and blunts renal autoregulatory behavior. We assessed afferent arteriolar autoregulatory behavior in male Sprague-Dawley rats after prolonged LPS (0.1 mg·kg-1·day-1 sq) infusion via osmotic minipump for 8 or 14 days. Some rats also received daily cotreatment with either anti-TLR4 antibody (1 μg ip), competitive antagonist peptide (CAP; 3 mg/kg ip) or tempol (2 mmol/l, drinking water) throughout the 8-day LPS treatment period. Autoregulatory behavior was assessed using the in vitro blood-perfused juxtamedullary nephron preparation. Selected physiological measures, systolic blood pressure and baseline diameters were normal and similar across groups. Pressure-dependent vasoconstriction averaged 72 ± 2% of baseline in sham rats, indicating intact autoregulatory behavior. Eight-day LPS-treated rats exhibited significantly impaired pressure-mediated vasoconstriction (96 ± 1% of baseline), whereas it was preserved in rats that received anti-TLR4 antibody (75 ± 3%), CAP (84 ± 2%), or tempol (82 ± 2%). Using a 14-day LPS (0.1 mg·kg-1·day-1 sq) intervention protocol, CAP treatment started on day 7, where autoregulatory behavior is already impaired. Systolic blood pressures were normal across all treatment groups. Fourteen-day LPS treatment retained the autoregulatory impairment (95 ± 2% of baseline). CAP intervention starting on day 7 rescued pressure-mediated vasoconstriction with diameters decreasing to 85 ± 1% of baseline. These data demonstrate that chronic subclinical TLR4 activation impairs afferent arteriolar autoregulatory behavior through mechanisms involving reactive oxygen species and major histocompatibility complex class II activation.