A method and apparatus for multi-cell calibration in a wireless communication system supporting cooperative Multiple Input Multiple Output (MIMO) are provided. Operations of a Base Station (BS) according to the method include transmitting a first message indicating a start of multi-cell sounding calibration to a reference Mobile Station (MS); estimating an uplink channel matrix of the reference MS using pilot symbols received from the reference MS; transmitting pilot symbols for enabling the reference MS to estimate a downlink channel matrix; when receiving downlink channel information of cooperative BSs from the reference MS, transmitting second messages comprising the downlink channel information to the cooperative BSs; and determining a multi-cell calibration matrix using the uplink channel matrix and the downlink channel information received from the reference MS.