Objective To characterize transmembrane protein 68 (TMEM68) in an alternative triacylglycerol (TAG) biosynthesis pathway, and determine the interplay between TMEM68 and the canonical TAG synthesis enzyme acyl-CoA:diacylglycerol acyltransferase (DGAT).Methods Effects of exogenous fatty acid and monoacylglycerol on TAG synthesis and lipid droplet (LD) formation in TMEM68 overexpression and knockout cells treated with DGAT inhibitor or not were investigated by comparing LD morphology, Oil Red O staining, and measurement of TAG levels. LDs were stained with fluorescence dye and observed by confocal fluorescence microscopy. TAG levels were determined with an enzyme-based triglyceride assay kit. Colocalization of TMEM68 and DGAT1 was detected by co-expression and confocal fluorescence microscopy and their interaction was determined by co-immunoprecipitation. RT-qPCR and immunoblotting assay were used to detect the expression of DGAT1.Results The synthesis of TAG catalyzed by TMEM68 was independent of DGAT activity. Surplus exogenous fatty acids and monoacylglycerol promoted TAG synthesis mainly through DGAT in human neuroblastoma cells. The LDs formed by TMEM68 were different in morphology from those by DGAT. In addition, TMEM68 and DGAT1 colocalized in the same endoplasmic reticulum (ER) compartment but did not interact physically. TMEM68 overexpression reduced the expression of DGAT1, the major DGAT enzyme involved in TAG synthesis, while TMEM68 knockout had little impact.Conclusion The TMEM68-mediated TAG synthesis pathway has distinct features from the canonical DGAT pathway, however, TMEM68 and DGAT may coregulate intracellular TAG levels.