A growing body of evidence suggests that p300 histone acetyltransferase plays important roles in cancer cell differentiation and proliferation. Here, we employed structure-based hierarchical virtual screening method to identify novel lead compounds of p300 histone acetyltransferase. From a screening library containing approximate 100 000 diverse druglike compounds, 33 compounds were chosen for experimental testing and one compound, 4-acetyl-2-methyl-N-morpholino-3,4-dihydro-2H-benzo[b][1, 4]thiazine-7-sulfonamide (17), showed as micromolar inhibitor. Based on its predicted binding pose, we investigated its binding characteristics by designing two series of structural modifications. The obtained structure-activity relationship results are consistent with the predicted binding model. We expect that the identified novel p300 histone acetyltransferase inhibitors will serve as starting points for further development of more potent and specific histone acetyltransferase inhibitors.