The invention relates to a rice anther culture improvement technology, and in particular relates to a method for improving the frequency of callus induction and plant differentiation rate in japonica rice anther culture. The method comprises the following steps that: 200g/667m<2> of 10% of paclobutrazol wettable powder and 50-60L of water are mixed for spraying treatment before sampling, the spraying of the paclobutrazol and low-temperature treatment cause multiple pollen cells in the anther to be in the later monokaryotic stage, novel cytokinin 4-FA is introduced into a callus inducing medium, under the synergistic action of the 4-FA, KT, and 2,4-D, division is easily started in the dark culture after inoculation, and the frequency of callus induction is greatly improved. By using the anther culture method, the frequency of callus induction in the japonica rice anther culture is obviously improved to exceed 85.0%. A differential medium comprises the following ingredients: an MS (Murashige and Skoog) basic culture medium, 0.5mg/L of NAA, 0.5mg/L of ZTO, 0.8mg/L of TDZ, 0.2mg/L of FA, 0.2mg/L of MET, 30g/L of saccharose, and 8g/L of agar powder; and after the novel hormones of ZT, 4-FA and TDZ are introduced, light cultivation is carried out, and plant differentiation rate can reach more than 86%.