Polo-like kinases play crucial roles throughout mitosis. We previously reported that wortmannin potently inhibits Polo-like kinase 1 (Plk1). In this study, we show that wortmannin also strongly inhibits Polo-like kinase 3 (Plk3). To further characterize this inhibition, we identified the sites of labeling on Plk1 and Plk3 targeted by AX7503, a tetramethylrhodamine-wortmannin conjugate. AX7503 labeling on Plk1 and Plk3 was found to occur on a conserved ATP binding site residue. In addition, we show that wortmannin inhibits Plk3 activity in live cells at concentrations commonly used to inhibit the more well known targets of wortmannin, the phosphoinositide 3-kinases. Importantly, we found that inhibition of Plk3 by wortmannin lead to a decrease in phosphorylation of p53 on serine 20 induced by DNA damage, demonstrating the effect of wortmannin on a downstream Plk3 target. Taken together, our results suggest that wortmannin can affect multiple functions of Plk3 in cell cycle progression and at the DNA damage check point. The identification of the labeling sites of Plk1 and Plk3 by AX7503 may be useful in designing more effective compounds to target Polo-like kinases for cancer treatment and also may be useful for the structural study of Plk domains.