Gallbladder cancer (GBC) is prone to lymph node metastasis. Lymph node (LN) metastasis is correlated with abysmal patient prognosis, but the underlying mechanism remains elusive. In this study, transcriptome sequencing of 6 paired GBC tumors and metastatic LNs was performed and identified eEF1A2 as key genes associated with GBC LN metastasis. qPCR, Western blotting and immunohistochemistry (IHC) were performed to assess the expression of eEF1A2 and relating proteins in GBC. The function of eEF1A2 and its regulators were demonstrated in different GBC cell lines as well as in xenograft models. Two independent cohorts of GBC patients were used to reveal the clinical significance. The results revealed that eEF1A2 is tightly correlated with lymph node metastasis and poor prognosis in patients with GBC. In two GBC cell lines, eEF1A2 knockdown impaired cell proliferation, migration, and invasion in vitro and inhibited tumor growth and lymph node metastasis in vivo, whereas overexpression of eEF1A2 promoted these processes. EEF1AKMT4 trimethylates eEF1A2 at K36 site in GBC and is essential for the tumor-promoting effect of eEF1A2. Mechanistically, trimethylation at the K36 site of eEF1A2 increased the GTPase activity of eEF1A2 and enhanced tumor promoting signals including ERK1/2 and AKT by promoting the ribosome total protein synthesis. In conclusion, the evolutionarily conserved EEF1AKMT4-eEF1A2K36me3-ribosome protein synthesis-tumor promoting signals axis acts as a mechanism that promotes GBC progression and may be a potential therapeutic target for GBC lymph node metastasis.