Abstract: To investigate the differential responses in huperzine A（Hup A） accumulation when different amino acids solutions were used for in vitro thallus culture of Huperzia serrata. Transcriptome sequencing was used for analysis of differentially expressed genes（DEGs） from thallis cultured with lysine and aspartic acid, and RT-qPCR was carried out to explore enzymes related to Hup A biosynthesis. Adding 1 mmol/L aspartic acid（D） could significantly promote the accumulation of Hup A, and its content was 84.05 μg/g dry weight（DW）, which was 1.29-fold than that of the control（65.15 μg/g DW）; the addition of 4 mmol/L lysine（K） solution significantly inhibited Hup A accumulation, and its content was 48.42 μg/g DW, which was 0.75-fold than that of the control. Transcriptome sequencing-bioinformatics alignment analysis of the aforementioned materials showed that in GO alignment analysis, functions were annotated for 16 258 unigenes. From the statistical analysis of the DEGs of the three groups, we found that there were 1 046, 782 and 1 586 DEGs for CK vs D, CK vs K, and D vs K, respectively, with D vs K having the most DEGs. DEGs that were enriched in KEGG metabolic pathways and validated by fluorescence quantitative PCR included PANK1, GDH2, APX, HA1, ND4 L, and COX1. The above results indicated that Hup A content and DEGs of thallus had significant different respone under the D and K treatments. Gene expression differences are the molecular basis that affects Hup A accumulation in in vitro thallus cultures. PANK1 and GDH2 encode enzymes that synthesize an alkaloid intermediate. Therefore, PANK1 and GDH2 may be key enzymes involved in the biosynthesis of Hup A precursors. This paper provided a technical method and molecular basis for the culture of H. serrata thallus in vitro to increase the yield of Hup A.