ABA-INSENSITIVE 3 (ABI3) has long been known for activation of storage protein accumulation. A role of ABI3 on oil accumulation was previously suggested based on a decrease of oil content in seeds of abi3 mutant. However, this conclusion could not exclude possibilities of indirect or pleiotropic effects, such as through mutual regulatory interactions with FUSCA3 (FUS3), an activator of oil accumulation. To identify that ABI3 functions independent of the effects of related seed transcription factors, we expressed ABI3 under the control of an inducible promoter in tobacco BY2 cells and Arabidopsis rosette leaves. Inducible expression of ABI3 activated oil accumulation in these non-seed cells, demonstrating a general role of ABI3 in regulation of oil biosynthesis. Further expressing ABI3 in rosette leaves of fus3 knockout mutant still caused up to 3-fold greater triacylglycerol accumulation, indicating ABI3 can activate lipid accumulation independently of FUS3. Transcriptome analysis revealed that LIPID DROPLET PROTEIN (LDP) genes, including OLEOSINs and CALEOSINs, were up-regulated up to 1000-fold by ABI3 in the absence of FUS3, while the expression of WRINKLED1 was doubled. Taken together, our results provide genetic evidence that ABI3 activates oil accumulation with or without FUS3, most likely through up-regulating LDPs and WRINKLED1.