Acylation Stimulating Protein (ASP), a lipogenic hormone, stimulates triglyceride (TG) synthesis and glucose transport upon activation of C5L2, a GPCR receptor. ASP-deficient mice have reduced adipose tissue mass due to increasing energy expenditure in spite of increased food intake. The objective of this study was to evaluate the blocking of ASP-C5L2 interaction via neutralizing antibodies (antiASP against ASP and antiC5L2-L1 against C5L2). In vitro, antiASP and antiC5L2-L1 blocked ASP binding to C5L2 and efficiently inhibited ASP stimulation of TG synthesis and glucose transport. In vivo, antiASP and antiC5L2-L1 did not alter body weight, adipose tissue mass, food intake, and hormone levels (insulin, leptin, and adiponectin), but they did induce a significant delay in TG clearance (p<0.0001, two-way RM ANOVA) and NEFA clearance (p<0.0001, two-way RM ANOVA). To evaluate the effects of neutralizing antibody treatment in tissues, TG mass, lipoprotein lipase (LPL), and AMP kinase (AMPK) activities were examined. In adipose tissue, there was no change in AMPK activity, but neutralizing antibodies decreased perirenal TG mass (-38.4% antiASP, -18.8% antiC5L2, p<0.01-0.001) and perirenal LPL activity (-75.6% antiASP, -72.5% antiC5L2, p<0.05). In liver, antiC5L2-L1 decreased TG mass (-42.8%, p<0.05) while antiASP increased AMPK activity (+34.6%, p<0.001). In the muscle, antiC5L2-L1 significantly increased TG mass (+128.0%, p<0.05), LPL activity (+226.1%, p<0.001), and AMPK activity (+71.1%, p<0.01). In addition, antiASP increased LPL activity (+164.4, p<0.05) and AMPK activity (+53.9%, p<0.05) in muscle. ASP/C5L2 neutralizing antibodies effectively block ASP-C5L2 interaction, altering lipid distribution and energy utilization.