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Pseuderanthemum palatiferum (Nees) Radlk. has been used extensively as a medicinal plant for the treatment of diabetes mellitus in various parts of Southeast Asia. However, the mechanism of its antidiabetic action has not been fully characterized yet. The objectives of this study were to investigate the in vitro effects of P. palatiferum leaf ethanolic extract (PPE extract) on adipocyte lipolysis, α-amylase enzyme activity and antioxidant activity. The PPE extract at every concentration tested (10, 25, 50, 100, 250, 500 µg/ml) caused a significant inhibition on basal lipolysis in adipocytes of the high fat diet-fed rats. The PPE extract also produced a concentration-dependent anti-lipolytic action against isoprenaline (0.1 µM)-induced lipolysis in the normal pellet diet-fed rat derived adipocytes. Inhibition of α-amylase was detected using the 3,5-dinitrosalicylic acid assay, the PPE extract having an IC 50 of 4.99 mg/ml. An antioxidant activity, as evaluated by the DPPH assay (IC 50), was 58.13 µg/ml. In the ferric reducing power assay, the extract exerted antioxidant activity with a vitamin C equivalent antioxidant capacity (VCEAC) and a trolox equivalent antioxidant capacity (TEAC) of 2.33 and 2.04 g/100 g extract, respectively. The anti-lipolytic, α-amylase inhibitory and antioxidant activities of PPE extract detected here help justify the traditional use of this plant in the treatment of diabetes mellitus.