Blunt thoracic trauma (TxT) is a common injury pattern in polytraumatized patients. When combined with a secondary trigger, TxT often results in acute lung injury (ALI), which negatively affects outcomes. Recent findings suggest that ALI is caused by both local and systemic inflammatory reactions. Club cell protein (CC)16 is an anti‑inflammatory peptide associated with lung injury following TxT. Recently, the anti‑inflammatory properties of endogenous CC16 in a murine model of TxT with subsequent cecal‑ligation and puncture (CLP) as the secondary hit were demonstrated by our group. The present study aimed to determine whether CC16 neutralization improves survival following 'double‑hit'‑induced ALI. For this purpose, a total of 120 C57BL/6N mice were subjected to TxT, followed by CLP after 24 h. Sham‑operated animals underwent anesthesia without the induction of TxT + CLP. CC16 neutralization was performed by providing a CC16 antibody intratracheally following TxT (early) or following CLP (late). Survival was assessed in 48 animals for 6 days after CLP. Sacrifice was performed 6 or 24 h post‑CLP to evaluate the anti‑inflammatory effect of CC16. The results revealed that CC16 neutralization enhanced pro‑inflammatory CXCL1 levels, thereby confirming the anti‑inflammatory characteristics of CC16 in this model. Early CC16 neutralization immediately following TxT significantly prolonged survival within 60 h; however, the survival rate did not change until 6 days post‑trauma. Late CC16 neutralization did not provide any survival benefits. On the whole, the present study demonstrated that neutralizing CC16 confirmed its anti‑inflammatory potential in this double‑hit ALI model. Early CC16 neutralization prolonged survival within 60 h; however, no survival benefits were observed after 6 days post‑CLP in any group.