<p>Jerusalem artichoke (<i>Helianthus tuberosus</i>) is highly valued as a source of essential nutrients and bioactive secondary metabolites (SMs) beneficial to human health. This study aims to establish an optimized in vitro callus culture system for Jerusalem artichoke and to evaluate the effects of explant type, callus texture, and methyl jasmonate (MeJA) elicitation on the production of total phenolics and flavonoids. The highest callus induction frequencies were achieved on medium supplemented with 0.5 mg L^− 1 6-benzylaminopurine (6-BA), 0.5 mg L^− 1 2,4-dichlorophenoxy acetic acid (2,4-D), and 0.25 mg L^− 1 thidiazuron (TDZ), with frequencies of 100.0% for leaf and stem explants and 97.0% for root explants. Maximum callus proliferation was recorded in the presence of 0.5 mg L^− 1 2,4-D and 1.0 mg L^− 1 TDZ. Anatomical observations revealed that compact calli were characterized by smaller cells with denser cytoplasm and more prominent nuclei compared to friable calli. Simple sequence repeat (SSR) characterization confirmed the genetic uniformity of both compact and friable calli. The accumulation of total phenolics and flavonoids was significantly influenced by both explant type and callus texture. Specifically, calli derived from stem transverse thin cell layers (tTCLs) exhibited higher levels of total phenolics, whereas root-derived calli demonstrated superior flavonoid production. Furthermore, compact calli accumulated significantly higher levels of these metabolites than their friable counterparts. Elicitation with 10 µM MeJA significantly increased total phenolic and flavonoid contents in the callus cultures by 2.18- and 2.29-fold, respectively, compared to the untreated control. These findings offer valuable insights for optimizing callus cultures and enhancing the production of valuable SMs in Jerusalem artichoke.</p>