A three-dimensional cell culture method for increasing cell proliferation efficiency by suitably regulating the proliferation-inducing and proliferation-inhibitory signals between cells is provided. The method includes repeatedly performing any one or both of the following processes a) and b) so as to regulate proliferation-inducing and proliferation-inhibitory signals between the cells: a) a process of gradually adding the micro-scaffolds, in which a small amount of the micro-scaffolds are used in an initial stage in order to maintain a suitable distance between the cells, and the amount of the micro-scaffolds is then increased slowly according to cell proliferation rate; and b) a periodic shaking process, in which shaking is performed in order to separate connected cells from each other by a physical force, after the cells are incubated for more than a given period of time.