Neuroblastoma (NB) is an aggressive cancer and has poor prognosis in children. TP-0903, a multi-kinase inhibitor, shows inhibitory effects on NB but the mechanistic act is not completely explored. Here, we aimed to explore the anticancer activity of TP-0903 against NB cells and its underlying mechanism. In this study, our findings showed that TP-0903 ( ≥ 50 nM) significantly inhibited the growth of SH-SY5Y and Neuro-2a cells. Further results revealed that TP-0903 remarkably triggered cell apoptosis, mitochondrial membrane potential (MMP) lose, and caspase activation. Microarray assay, qRT-PCR, and Western blotting results indicated that DKK1 was downregulated by TP-0903. Notably, DKK1 is upregulated in NB tissues as comparing to normal tissues. Moreover, silencing DKK1 promoted TP-0903-induced apoptosis and caspase activation, and predicted the binding of TP-0903 to DKK1. In addition, we found that 3'-UTR of DKK1 had a potential target region for miR-335-3p and TP-0903 upregulated miR-335-3p expression. Of important, miR-335-3p mimic combined with TP-0903 provoked higher apoptosis and caspase activation than TP-0903 alone. We also observed that TP-0903 increased cellular reactive oxygen species (ROS), and inhibition of ROS reduced the apoptosis, PARP cleavage, and miR-335-3p, while increasing DKK1 in response to TP-0903. Finally, we demonstrated that TP-0903 significantly diminished the tumor growth and DKK1 expression in xenograft mice. Collectively, our findings indicate that TP-0903 triggers apoptotic cell death of NB cells, attributing to the ROS-mediated miR-335-3p upregulation and the consequent DKK1 downregulation.