Multiple acyl-CoA deyhydrogenase deficiency (MADD) is caused by defects in electron transfer flavoprotein and ETF-ubiquinone oxidoreductase (ETF-QO). Recently, a common sequence variant in ETFDH, encoding ETF-QO, was initially identified in Taiwanese MADD patients with carrier frequency about 1%. Several subsequent reports supported that this probable founder mutation, c.250G > A (p.Ala84Thr), is the most common cause of MADD in southern Chinese population. Later-onset MADD clinically present with episodic muscle weakness under acute metabolic decompensation, cause mortality due to metabolic crisis and pathologically lead to intracellular lipid accumulation. Riboflavin supplementation has shown its efficacy in the MADD patients, particularly with ETFDH mutations and later onset form. As MADD may be the most common hereditary disorder of lipid metabolism in southern Chinese population and it is potentially treatable, we used different analyses in vitro to show the pathogenesis of MADD caused by this common mutation, c.250G > A in ETFDH, including assessments of lipid and acylcarnitines accumulation, neurite outgrowth and reactive oxygen species (ROS) production. We successfully observed increased intracellular lipid droplets, elevated acylcarnitine concentration, neurite shortening and augmented ROS production in transfected cells which recapitulates the pathological and biochemical phenotypes in human. These results prove that this hot spot mutation is a true pathogenic mutation, not a polymorphism. Screening this hot spot mutation for Chinese patients clinically suspected to have MADD would be helpful for early diagnosis of MADD for which riboflavin supplementation may be effective. In addition, this in vitro system may be also useful for screening potential therapeutic strategies and further exploring the pathomechanism of MADD.