Inflammation is involved in ventilator-induced diaphragm dysfunction. Toll-like receptor 4 (TLR4) is an important inflammatory factor, but it remains unclear whether TLR4 contributes to ventilator-induced diaphragm dysfunction. This study aimed to investigate the role of TLR4 signaling in ventilator-induced diaphragm dysfunction. Total 30 adult male SD rats were randomly divided into control group, low tidal volume and high tidal volume group (n = 10). Control group received tracheotomy and endotracheal intubation but no mechanical ventilation; low tidal volume group and high tidal volume group received tracheotomy, mechanical ventilation after intubation, and then received tidal volume 6 ml/kg and 20 ml/kg, respectively. Ventilation rate was 60 beats/min, inspiratory to expiratory ratio was 1:3, FiO2 was 21%, ventilation was 24 h. Diaphragmatic muscle contraction, tumor necrosis factor (TNF-α) and TLR4 expression, and malondialdehyde (MDA) and superoxide dismutase (SOD) contents in the diaphragm tissues were detected. TLR4 and TNF-α expression in diaphragm tissues of high tidal volume group were significantly increased and diaphragm muscle contraction was significantly decreased, compared to other groups. In conclusion, high tidal volume ventilation may activate TLR4 signaling and cause pathological changes in diaphragm tissues. TLR4 is a promising target for the prevention and treatment of ventilator-associated diaphragmatic injury.