Background: Banisteriopsis pubipetala is a little‑studied species belonging to the same genus of B. caapi, which stands out for the bioactive compounds known to be important in the degenerative diseases treatment. Objective: The objective is to analyze the anti‑inflammatory and cytotoxic potential and chromatographic profile of the extracts of B. pubipetala. Materials and Methods: The investigation of the chromatographic profile was performed through high‑performance liquid chromatography diode‑array detector. Cell viability was determined by a quantitative colorimetric assay with 3‑(4,5‑dimethylthiazol‑2yl)‑2,5‑diphenyltetrazolium bromide. In the pharmacological tests, interleukin (IL)‑6, tumor necrosis factor‑alpha (TNF‑α), IL‑10, and nitric oxide (NO) levels in cell culture supernatants were performed. Results: In the leaf were observed nine major compounds and in the stem five major compounds. The extracts of B. pubipetala demonstrated concentration‑dependent behavior regarding cytotoxicity. The fraction in dichloromethane had inhibitory concentration of 50% (IC50) = 67.39 μg/mL whereas the extract in ethyl acetate had IC50 = 103.37 μg/mL. The results showed that extracts significantly reduced the production of IL‑6 and TNF‑α by cells 3T3 cells, however increased the production of IL‑10 and NO. Conclusion: The results of the tests indicate that the extracts of B. pubipetala evaluated have potential anti‑inflammatory properties and may promote the regulation of inflammation levels.