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Massive multiple-input multiple-output (MIMO) technology obtains better transmission efficiency because of the number of antennas at transmitting end and receiving end increased up to a few hundred, but it will also limit the performance due to pilot contamination arise from interference among users. In order to get the pilot contamination problem in massive MIMO systems settled, an Ant Colony Optimization Pilot Assignment strategy is proposed. Firstly, it uses the interference path diagram among users according to the user's channel transmission characteristics. Secondly, it performs optimization iterations according to the idea of the ant colony optimization algorithm to find the optimum route with the least total interference among users. Furthermore, it performs pilot assignment to users according to the required path to obtain a pilot assignment scheme with the least interference among users. The simulation results prove that the proposed method can suppress the pilot contamination problem and improve the performance of communication system with advantage.