Directed neuronal differentiation is crucial for development of cell therapy and investigation of neurogenesis. However, limited differentiation-inducing agents are available and most of current differentiation regimens are complicated. Here we carried out a combinatorial high-throughput screen with a random siRNA library on murine P19 cell differentiation toward neuronal lineage. Two siRNAs screened from the library were able to direct neuronal differentiation, determined by nestin, neurofilament-M and MAP-2 up-regulation. This is the first trial of a screening procedure for neuronal differentiation-directing agents arising from a random siRNA library and demonstrates that a random siRNA library can be considered as a new resource in efforts to seek new agents for directed differentiation. As the random siRNA library has a broad coverage for the entire genome, screening using random siRNA library can be expected to greatly augment the repertoire of siRNAs for directed differentiation.