Myristic acid, the 14-carbon saturated fatty acid (C14:0), is usually associated with negative consequences for human health, and in particular its consumption is correlated to an increased cardiovascular disease risk. Since it is a little abundant into the cells, its specific properties and functional roles have not been fully described. The aim of this study was to explore the cell response to this fatty acid to help explain clinical findings on the relationship between C14:0 and cardiovascular disease. The human liver HepG2 cell line was used as a model to investigate the hepatic response to C14:0 in a combined proteomic and secretomic approach. A total of 47 intracellular and 32 secreted proteins were found to be deregulated among the different dosages of C14:0 treatments (50, 125, and 250 μM) as compared to control. Bioinformatics analysis revealed that myristic acid modulates the lipid droplet formation and the cytoskeleton organization, in addition it induces ER stress as well as changes in exosome and extracellular miRNA sorting. Our data provide for the first time quantitative proteomic data regarding C14:0 and contribute to the explanation of the molecular mechanisms through which this fatty acid can cause adverse health effects.