Disruption of acylcarnitine homeostasis results in life-threatening outcomes in humans. Carnitine– acylcarnitine translocase deffciency (CACTD) is a scarce autosomal recessive genetic disease and may result in patients’ death due to heart arrest or respiratory insufffciency. However, the reasons and mechanism of CACTD inducing respiratory insufffciency have never been elucidated. Herein, we employed lipidomic techniques to create comprehensive lipidomic maps of entire lungs throughout both prenatal and postnatal developmental stages in mice. We found that the acylcarnitines manifested notable variations and coordinated the expression levels of carnitine–acylcarnitine translocase (Cact) across these lung developmental stages. Cact-null mice were all dead with a symptom of respiratory distress and exhibited failed lung development. Loss of Cact resulted in an accumulation of palmitoyl-carnitine (C16-acylcarnitine) in the lungs and promoted the proliferation of mesenchymal progenitor cells. Mesenchymal cells with elevated C16-acylcarnitine levels displayed minimal changes in energy metabolism but, upon investigation, revealed an interaction with sterile alpha motif domain and histidine-aspartate domain-containing protein 1 (Samhd1), leading to decreased protein abundance and enhanced cell proliferation. Thus, our ffndings present a mechanism addressing respiratory distress in CACTD, offering a valuable reference point for both the elucidation of pathogenesis and the exploration of treatment strategies for neonatal respiratory distress.