Penehyclidine hydrochloride (PHC) is a selective M₁ and M₃ receptor antagonist. This study was designed to investigate the effect of PHC on acute lung injury (ALI) induced by severe acute pancreatitis (SAP) and the expression of hypoxia-inducible factor-1α (HIF-1α) in rats. A total of 45 healthy adult male SD rats were randomly divided into 3 groups: an S group, sham operation; an ALI group, pancreatitis-associated acute lung injury (PALI); and a P group, PALI treated with PHC. Rats from the ALI and P groups were used to establish a model of acute lung injury associated with SAP by retrograde injection of 4% sodium taurocholate into the biliopancreatic duct. Rats in the P group, reflecting acute lung injury caused by SAP, were treated with PHC immediately following SAP. Rats in the S and ALI groups were injected with the same amount of 0.9% sodium chloride solution. After modeling, the rats were sacrificed at 12h. The wet/dry weight (W/D) ratios of lung tissue were calculated. Pathological changes in pancreatic and lung tissues were scored. The expression levels of TLR4 and NF-κB p65 in lung tissue were detected by Western blot. RT-PCR was used to detect HIF-1α mRNA in lung tissue. The HIF-1α, IL-1β, and IL-6 expression levels in lung tissues and serum amylase levels were detected by ELISA. The results showed extensive infiltration of neutrophils, alveolar hemorrhage and necrosis and fat necrosis in the pancreatic tissue of rats in the PALI and P groups. Their pancreatic tissue injury scores were significantly higher than the score of the S group (P<0.01). However, no statistically significant difference was observed in the serum amylase levels of the P and ALI groups (P>0.05). The W/D ratios of lung tissue in the ALI and P group rats were significantly higher than those in the S group (P<0.05). Compared with those of the ALI group rats, the lung tissue pathological changes of the P group were significantly improved, and the lung W/D value was significantly lower than that of the ALI group (P<0.05). Compared with those of the S group, the TLR4, NF-κB p65, HIF-1α mRNA, and HIF-1α expression levels in the lung tissue of the ALI and P groups were significantly higher (P<0.01), and the TLR4, NF-κB p65, HIF-1α mRNA, HIF-1α, IL-1β and IL-6 expression levels in the P group were significantly lower than those in the ALI group (P<0.05). The current work indicates that PHC could not alleviate the damage to pancreatic tissue caused by SAP. However, PHC did suppress HIF-1α, IL-1β and IL-6 expression levels and reduced the acute lung injury induced by SAP in rats, which might depend on suppression of the expression of inflammatory factors, such as HIF-1α.