Indigofera truxillensis Kunth is traditionally used in Brazilian folk medicine as an oral infusion of the aerial parts, taken as one cup several times daily for at least one week, to relieve inflammatory pain and gastrointestinal complaints. To chemically characterize and evaluate the anti-arthritic and analgesic properties of the methanolic extract (MEIT) and indigo obtained from the aerial parts of I. truxillensis. The chemical analysis of MEIT and the identification of indigo were performed by electrospray ionization tandem mass spectrometry and nuclear magnetic resonance. Anti-inflammatory and anti-hyperalgesic activities were assessed in mice using carrageenan-induced pleurisy and paw inflammation, complete Freund's adjuvant (CFA), and zymosan-induced articular inflammation models. Cytotoxicity and phagocytic activity were evaluated in vitro using leukocyte viability (MTT) and neutrophil phagocytosis assays. MEIT and indigo significantly inhibited leukocyte recruitment in carrageenan-induced pleurisy. In the carrageenan paw model, both reduced mechanical hyperalgesia, cold sensitivity, and edema. In CFA-induced inflammation, MEIT and indigo decreased mechanical hyperalgesia, cold response, myeloperoxidase activity, and protein exudation. In the zymosan-induced arthritis model, both treatments reduced knee edema, mechanical hyperalgesia, leukocyte migration, protein extravasation, and nitric oxide levels in synovial fluid. MEIT and indigo did not exhibit cytotoxicity in vitro; MEIT reduced neutrophil phagocytosis at specific concentrations, whereas indigo did not alter phagocytic activity. MEIT and indigo exhibit significant anti-inflammatory, anti-hyperalgesic, and anti-arthritic activities, supporting the traditional use of I. truxillensis for painful and inflammatory conditions and identifying indigo as a key bioactive constituent.