To understand protein S-palmitoylation's role in macrophage reprogramming, dynamic changes were comprehensively mapped in immortalized mouse macrophages during early LPS stimulation using acyl-biotin exchange (ABE) proteomics enhanced with multi-protease digestion. This approach identified 2613 putative S-palmitoylated proteins, including 587 novel mouse candidates, many involved in immunity. Following 30 minutes of LPS exposure, 648 proteins showed altered palmitoylation, indicating dynamic regulation linked functionally to immune signaling, transport, cellular organization, and metabolism. This research provides a key resource on the macrophage palmitoylome's remodeling during immune activation, uncovering new potential regulatory targets.