Aim Little is known about how toll‐like receptor 4 (TLR4) influences the renal microvasculature. We hypothesized that acute TLR4 stimulation with lipopolysaccharide (LPS) impairs afferent arteriole autoregulatory behaviour, partially through reactive oxygen species (ROS). Methods We assessed afferent arteriole autoregulatory behaviour after LPS treatment (1 mg kg−1; i.p.) using the in vitro blood‐perfused juxtamedullary nephron preparation. Autoregulatory behaviour was assessed by measuring diameter responses to stepwise changes in renal perfusion pressure. TLR4 expression was assessed by immunofluorescence, immunohistochemistry and Western blot analysis in the renal cortex and vasculature. Results Baseline arteriole diameter at 100 mmHg averaged 15.2 ± 1.2 μm and 12.2 ± 1.0 μm for control and LPS groups (P < 0.05) respectively. When perfusion pressure was increased in 15 mmHg increments from 65 to 170 mmHg, arteriole diameter in control kidneys decreased significantly to 69 ± 6% of baseline diameter. In the LPS‐treated group, arteriole diameter remained essentially unchanged (103 ± 9% of baseline), indicating impaired autoregulatory behaviour. Pre‐treatment with anti‐TLR4 antibody or the TLR4 antagonist, LPS‐RS, preserved autoregulatory behaviour during LPS treatment. P2 receptor reactivity was normal in control and LPS‐treated rats. Pre‐treatment with Losartan (angiotensin type 1 receptor blocker; (AT1) 2 mg kg−1; i.p.) increased baseline afferent arteriole diameter but did not preserve autoregulatory behaviour in LPS‐treated rats. Acute exposure to Tempol (10−3 mol L−1), a superoxide dismutase mimetic, restored pressure‐mediated vasoconstriction in kidneys from LPS‐treated rats. Conclusion These data demonstrate that TLR4 activation impairs afferent arteriole autoregulatory behaviour, partially through ROS, but independently of P2 and AT1 receptor activation.