Stress stimulates sympatho‐adrenomedullary activity and lipolysis in adipocytes. Increase in plasma free fatty acids has been demonstrated to be a major link between obesity and insulin resistance. However, there are many reports describing the anti‐obesity effect of estrogen. This study investigated the effect of estrogen on isoproterenol (ISO) activated lipolysis in murine adipocytes. ISO (5 μM) and 17β‐Estradiol (E2) was added to mature 3T3‐L1 adipocytes in 6 well plates. After 2 hr incubation at 37 °C in 5% CO2/95% air, the glycerol concentration in DMEM medium was measured. Effects of β2‐adrenoceptor inhibitor (yohimbine, 50 μM), phosphodiesterase inhibitor (cilostamide, 2 μM) and estrogen receptor blocker (tamoxifen, 10 μM) on the inhibitory effect of lipolysis of E2 were also tested. Further, effect of E2 on cAMP production and hormone sensitive lipase (HSL) activity in 3T3‐L1 adipocytes were investigated. E2 (5–50 μM) inhibited ISO activated lipolysis in 3T3‐L1 adipocytes dose‐dependently. Yohimbine, cilostamide and tamoxifen did not abolish the inhibitory effect of E2 on ISO activated lipolysis. E2 inhibited HSL activity, whereas E2 did not reverse the increased cAMP level induced by ISO stimulation. These results suggest that E2 exerts its inhibitory effect of ISO activated lipolysis by inhibition somewhere down‐stream of cAMP production but up‐stream of the HSL signaling pathway.