This paper introduces an improved frequency domain channel estimation method based on interpolation vectors for single carrier frequency domain equalization (SC-FDE) with the multiple-input multiple-output (MIMO) scheme. The proposed algorithm is derived by employing the least squares (LS) criterion, and a specified application for the wide sense stationary uncorrelated scattering (WSSUS) Rayleigh fading channel is presented. The channel frequency domain responses estimated at two adjacent pilot blocks are used to track the time-variant channel information, which can effectively improve the accuracy of channel estimation without significantly increasing complexity. Maximum mean square error (MMSE) frequency domain equalization based on the estimated channel is employed in the receiver to recover transmitted signals. This paper also investigates a training sequence design method for multiple transmit antennas and a noise variance estimation method. Numerical simulation results show that the proposed methods can perform very well for fading channels with long multipath delay and high Doppler spread.