Rhizoma et Radix Notopterygii is often used as a traditional medicine in China. In our recent work, we found that the ethanol crude extract of Rhizoma et Radix Notopterygii could reduce phytotoxicity of acetochlor on rice. The crude extract of Rhizoma et Radix Notopterygii was isolated and purified by activity-guided fractionation. Two coumarins, isopimpinellin and 5-methoxypsoralen (5-MOP) were identified, and their bioactivity was tested in a growth chamber. The results showed that the two coumarins increased herbicide tolerance of rice shoots, and 5-MOP demonstrated better protection than isopimpinellin. The treatment of 5-MOP at 50 g ai ha−1 recovered the shoot height of rice from 42.6% (treated with acetochlor only) to 81.6% of the control treated without acetochlor, whereas the phytocidal activity of acetochlor on barnyardgrass was not impaired by 5-MOP. Further study suggested that 5-MOP increases herbicide tolerance of rice by enhancing the glutathione S-transferase level of activity in rice. Our findings suggest that isopimpinellin and 5-MOP have the potential to be applied as safeners for rice.