Background Interleukin 35 (IL35) has been reported to play a role in acute lung injury (ALI); however, the current results regarding the relationship between IL35 and ALI are inconsistent. Therefore, we aimed to further determine the function of IL35 in ALI in mice and the potential mechanism in this paper. Materials and methods Hematoxylin-eosin (HE) staining and Masson staining were used to evaluate lung injury in mice. Immunohistochemical staining was used to evaluate the expression of IL35 p35, TLR4 and MD2 and the Bax/Bcl2 and p-P65/P65 ratios. The expression levels of IL35 EBi3, CD68, CD206 and MPO were assessed by immunofluorescence staining. RT-PCR was used to examine the expression levels of IL1 beta and IL6. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was performed to detect apoptotic cells. Results Overexpression of IL35 alleviated LPS-induced ALI in mice. IL35 overexpression decreased the expression of CD68 and increased the expression of CD206 in mice with ALI. Furthermore, upregulation of IL35 expression obviously reduced the expression of MPO, IL1 beta and IL6 in the lung tissues of mice with ALI. Mechanistically, IL35 suppressed the TLR4/NF kappa B-P65 pathway, leading to the promotion of the M1 to M2 macrophage transition and alleviation of inflammation in mice with ALI. Conclusions IL35 relieved LPS-induced inflammation and ALI in mice by regulating M1/M2 macrophage polarization and inhibiting the activation of the TLR4/NF kappa B-P65 pathway.