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Anthocyanins biosynthesis was regulated by MYB-bHLH-WD40 protein complex at transcriptional level.Previous results showed regulation of the anthocyanin biosynthesis in litchi by LcMYB 1,but basic helix-loophelix proteins(bHLHs)as partners had not been identified yet.The present study describes the functional characterization of three litchi bHLH candidate anthocyanin regulators,LcbHLH1,LcbHLH2 and LcbHLH3.Although these three litchi bHLHs phylogenetically clustered with bHLH proteins related to anthcoyanin biosynthesis in other plant,only LcbHLH1 and LcbHLH3 were found to localize in the nucleus and to physically interact with LcMYB1.None of these bHLHs transcription level coordinated with anthocyanin accumulation in different tissues and during development.However,when co-infiltrated with LcMYB1,both LcbHLH1 and LcbHLH3 enhanced anthocyanin accumulation in tobacco leaves with LcbHLH3 being the best inducer.Significant accumulation of anthocyanin in leaves transformed with the combination of LcMYB1 and LcbHLH3 involves the up-regulation of two tobacco endogenous bHLH regulators,NtAN1a and NtAN1b and late structural genes,like NtDFR and NtANS.Significant activity of the ANS promoter was observed in transient expression assays either with LcMYB1-LcbHLH1 or LcMYB1-LcbHLH3,while only minute activity was detected after transformation with only LcMYB1.Instead no activity was measured after induction with the combination of LcbHLH2 and LcMYB1.LcbHLH1 and LcbHLH3 are essential partner of LcMYB1 in regulating the anthocynin production in tobacco and probably also in litchi.The LcMYB1-LcbHLH complex enhanced anthocyanin accumulation may associate with activating the transcription of DFR and ANS.