Cancer, the second major cause of mortality trailing the cardiovascular diseases, is a multifactorial heterogeneous disease and growing public health problem worldwide. Owing to severe adverse effects of currently available therapies, there is a growing interest in natural compounds present in our daily diet. Among various natural products, flavonoids-polyphenolic compounds have attracted much attention and have been well-documented for their biological activities. Some flavonoids may inhibit cancer cell proliferation by modulating the action of different enzymes and signal transduction pathways. In the present study, we have evaluated the effect of pinostrobin, a natural flavonoid, on the catalytic activity of topoisomerase I, an essential enzyme for normal DNA replication. Catalytic inhibition of topoisomerase I activity would impair DNA replication of rapidly dividing cancer cells and hence inhibits tumor progression. Pinostrobin interaction with the topoisomerase I and DNA assessed in silico indicated it to form a ternary complex with both. In silico data also suggested pinostrobin to be an effective allosteric inhibitor for topoisomerase I. Further, in vitro investigations such as ethidium bromide displacement assay and spectroscopic studies supported in silico results on the binding of pinostrobin at the interface of topoisomerase I and DNA. Pinostrobin effectively inhibited topoisomerase I activity in vitro further confirming our in silico and in vitro findings. Since topoisomerase I is essential for DNA replication, inhibition of its activity by pinostrobin highlights the therapeutic potential of pinostrobin as an anti-proliferative agent.