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A new learning method of radial basis function (RBF) network based on fuzzy C-means (FCM) clustering and ant colony optimization (ACO) is presented in the paper. Generally, the crucial problem of learning RBF network is the selection of the unit number, the centers and widths of the Gaussian radial basis function in hidden layer, and the weights between hidden layer and output layer. In this work, the centers are determined by FCM clustering, and the weights are determined by least mean squares (LMS) method. In addition, ACO is introduced to optimize two important parameters of FCM including the weighting exponent which impacts its performance, and the clustering number that is equal to the hidden unit number which impacts the generalization of RBF network, by minimizing an objective function integrated the generalization error with the hidden unit number of RBF network. Simulation results of identifying a nonlinear system illustrate the effectiveness of designing the RBF network with smaller structure but stronger generalization ability, comparing with K-means and orthogonal least squares (OLS) based learning methods.