Background: Melothria heterophylla (family: Cucurbitaceae), commonly known as kudari, is used in the Indian traditional medicine to treat various inflammation-associated diseases, such as asthma, arthritis and pain. However, the anti-inflammatory active components of this plant have not been identified yet. The aim of this study was to investigate the potential analgesic and anti-inflammatory activities of a compound, quercetin-3-methoxy-4′-glucosyl-7-glucoside, isolated from M. heterophylla. Methods: The anti-inflammatory activity was determined using carrageenan- and dextran-induced rat paw edema as well as cotton pellet-induced granuloma in rats, whereas the analgesic activity was analyzed using acetic acid-induced writhing, hot plate and tail flick response in mice. The test compound was orally administered at a dose of 5, 10 or 15 mg/kg. The cyclooxygenase-1 (COX-1)- and COX-2-inhibitory capacity of the test compound was studied by enzyme immunosorbent assay. Results: Quercetin-3-methoxy-4′-glucosyl-7-glucoglucoside at 15 mg/kg exhibited a maximum inhibition of carrageenan-induced inflammation (50.3%, p < 0.05), dextran (52.8%, p < 0.05), and cotton pellets (41.4%, p < 0.05) compared to control animals. At the same dose, it showed a 73.1% inhibition (p < 0.05) of the pain threshold in acetic acid-induced writhing model. It also exhibited a considerable analgesic activity by prolonging the reaction time of the animals based on hot plate as well as tail flick response. The test compound was found to inhibit COX-1 (IC50 2.76 µg/mL) and more efficiently, COX-2 (IC50 1.99 µg/mL). Conclusions: Quercetin-3-methoxy-4′-glucosyl-7-glucoside possessed substantial analgesic and anti-inflammatory activities possibly due to inhibition of prostaglandin production, supporting the ethnomedicinal application of M. heterophylla to treat various inflammatory disorders.