Palmitic acid (C16:0) is the most abundant saturated fatty acid in animals serving as a substrate in synthesis and β-oxidation of other lipids, and in the modification of proteins called palmitoylation. The influence of dietary palmitic acid on protein S-palmitoylation remains largely unknown. In this study we performed high-throughput proteomic analyses of a membrane-enriched fraction of murine liver to examine the influence of a palm oil-rich diet (HPD) on S-palmitoylation of proteins. HPD feeding for 4 weeks led to an accumulation of C16:0 and C18:1 fatty acids in livers which disappeared after 12-week feeding, in contrast to an accumulation of C16:0 in peritoneal macrophages. Parallel proteomic studies revealed that HPD feeding induced a sequence of changes of the level and/or S-palmitoylation of diverse liver proteins involved in fatty acid, cholesterol and amino acid metabolism, hemostasis, and neutrophil degranulation. The HPD diet did not lead to liver damage, however, it caused progressing obesity, hypercholesterolemia and hyperglycemia. We conclude that the relatively mild negative impact of such diet on liver functioning can be attributed to a lower bioavailability of palm oil-derived C16:0 vs. that of C18:1 and the efficiency of mechanisms preventing liver injury, possibly including dynamic protein S-palmitoylation.