Lipid profiling of human plasma by liquid chromatography-electrospray ionization coupled to mass spectrometry (LC–ESI-MS) is being used to identify biomarkers of health, disease, and treatment efficacy. However, there is no consensus on the choice of anticoagulant to perform and compare lipidomic measurements. This study assessed the effect of the anticoagulants citrate, EDTA, and heparin, on eight synthetic and 80 plasma lipids, and compared lipidomic data among anticoagulants. Lipid extraction was affected distinctively by the anticoagulant of choice likely due to the different physicochemical properties among anticoagulants. Peak areas of seventy endogenous lipids showed significant differences between citrate–heparin and EDTA–heparin comparisons similar to those observed for synthetic lipids. Only ten endogenous lipid species showed comparable peak areas among the three anticoagulants. Correction by a structurally related internal standard only partly eliminated differences among anticoagulants (ANOVA, P value \0.001). However, comparisons among anticoagula were possible for most endogenous lipids after correction of peak areas by the sum of areas of its lipid class. Our observations indicate that the choice of anticoagulant distinctively impact the peak response of most lipid species by LC–ESI-MS. Lipidomic data from plasma obtained with different anticoagulants should address differences in matrix effects and extraction procedures since ion strength, plasma pH, and different physicochemical properties among anticoagulants influence lipid extraction and LC–ESI-MS analysis.