Artemisinin, an antimalarial secondary metabolite produced in Artemisia species, also has been recognized as an allelochemical that inhibits the growth of several plant species. However, the phytotoxicity mechanism of artemisinin is not exhaustively deciphered up to now. In this research, the effects of artemisinin on Arabidopsis thaliana root gravitropic curvature and development were characterized. Exogenously applied artemisinin disturb the root gravitropic responses, inhibited the elongation of primary and lateral roots and root hairs in a concentration-dependent fashion, and prevented the formation of lateral roots and root hairs. Moreover, the number of starch grain and the distribution range of auxin in the root tip was reduced by artemisinin, and the redistribution of auxin was less sensitive to gravity stimulus when treated with artemisinin than that of control. The expression of auxin transporter PIN2 was partially suppressed by artemisinin. Together, the results demonstrated that the effects of artemisinin on root gravitropism and root system development were largely dependent on the reduction of starch grain and auxin levels, as well as the disordered lateral auxin redistribution.