Acute lung injury (ALI) is a common respiratory syndrome accompanied with an inflammation response. Annexin A5 (AnxA5) has anti-thrombotic, anti-apoptotic, and anti-inflammatory properties. The current study aims to explore the potential effect of AnxA5 on lipopolysaccharide (LPS)-induced inflammatory response in alveolar macrophages (AMs). Rat AMs (NR8383) were used in this study, and the cell viabilities at 4, 8, and 16 h after LPS administration with gradient concentrations were determined using cell counting kit-8 assay. Cell apoptosis and expressions of messenger RNAs (mRNAs) and protein were determined by flow cytometry, quantitative real-time polymerase chain reaction (qRT-PCR), and western blot, respectively. We found that LPS suppressed the viability of AMs in a dose-dependent manner, and it elevated the expression of AnxA5 in AMs. Inhibition of AnxA5 improved the cell viability compared with the LPS group and could reduce the apoptosis rate in comparison with LPS treatment. The knockdown of AnxA5 suppressed the expressions of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL-1 beta), and IL-6 at both protein and mRNA levels and regulated the expressions of apoptosis-related molecules (Bax, Bcl-2, and caspase-3). Moreover, the knockdown of AnxA5 improved the expression levels of inhibitory kappa B (I kappa B) and nuclear factor E2-related factor 2 (Nrf2) but inhibited the expression of nuclear transcription factor kappa B (NF-kappa B), compared with the LPS group. SN50 and ML385 were used to validate this signaling, and the inhibition of AnxA5 suppressed the LPS-induced inflammation, indicating that AnxA5 may be a potential anti-inflammatory target. In addition, NF-kappa B/Nrf2 signaling pathway may also be involved in the LPS-induced inflammatory response of rat alveolar macrophages.