Sphaeralcea angustifolia is used in Mexican traditional medicine to treat inflammatory processes. SCopoletin (SC), TOmentin (TO), and sphaeralcic acid (SA) were reported as the main anti-inflammatory compounds in this species. The aim of this study was to establish in vitro conditions for the development of calli and cell suspension cultures that are the producers of these active compounds. Callus cultures of plant leaf explants were set up using different auxin levels of α-naphthalene acetic acid (NAA) in combination with a constant concentration (0.1 mg L−1) of Kinetin (Kn) in Murashige and Skoog (MS) medium. Optimal combinations for callus induction were 1.0 and 2.0 mg L−1 of NAA. SC, TO, and SA were not detected in callus tissues. Employing a 4 % inoculum in fresh biomass, cell suspension was established from friable callus with 1.0 mg L−1 of NAA in combination with 0.1 mg L−1 of Kn in MS liquid medium (27.4 mM nitrate). The cellular suspension synthesized SC and SA, SC was excreted into the culture medium, while SA was excreted into the culture medium and accumulated in biomass. To improve SC and SA production, total nitrate content was reduced in MS medium. On diminishing nitrate content to 2.74 mM, cellular suspension growth was not modified. SC concentration (0.04 %) was 60-fold higher than that detected in the wild plant (0.00067 %), TO was produced (0.096 %), and SA content (0.0036 %) was not improved. SA production in MS medium with 0.274 mM nitrate (0.004 %) was enriched 12-fold (0.0003 %) in relation to that of the wild plant. The anti-inflammatory effects at 5 h of intraperitoneal (i.p.) administration (100 mg per kg BW) of dichloromethane extracts from the medium (42 ± 3 %) and biomass (39 ± 9.3 %) of S. angustifolia cell suspensions cultivated in MS with 2.74 mM nitrate were similar. The effect of the biomass dichloromethane extract was dose dependent with a median Effective Dose (ED50) of 137.63 mg per kg BW.