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Callus growth and development, a crucial process in plant propagation, is involved in hormonal balance and abundant gene regulation. MiRNAs are key regulators in the process of cell differentiation and development. MiR397 was identified as participating in plant growth, development, and response to stress, and it was regulated by targeting the <i>LAC</i> gene. The regulatory function of miR397 during callus growth and development was not clear in <i>Liriodendron</i>. In this study, LhmiR397a and its targets were identified, and its regulatory function between LhmiR397a and <i>LhLAC11</i> was shown using qRT-PCR and transient expression in protoplasts. Furthermore, to clarify the regulatory function of LhmiR397a-<i>LhLAC11</i>, transgenic calli overexpressing <i>LhMIR397a</i>, <i>LhLAC11</i>, and <i>mLhLAC11</i> were separately obtained by <i>Agrobacterium</i>-mediated transfer. The results showed that overexpressing <i>LhMIR397a</i> might retard callus proliferation, while overexpressing LhLAC11 or mLhLAC11 could promote callus proliferation. Genes associated with the cell cycle had decreased expression when LhMIR397a was overexpressed, while increased expression was observed when LhLAC11 or mLhLAC11 was overexpressed. Additionally, the calli overexpressed with <i>LhMIR397a</i> could generate early cotyledons 21 days after induction, and the somatic embryo induction time was short compared with other genotypes. This study identified LhmiR397a and its targets and provided a functional characterization of LhmiR397a in callus growth and development by regulating its target in <i>Liriodendron</i>.