Rice seedling chlorosis is a common symptom in calcareous soil under drip irrigation,limiting rice growth and yield. A two-factor split-plot experiment was carried out to reveal the effects of sowing date and soil acidification on rice chlorosis caused by iron deficiency under drip irrigation with plastic film mulching(DIPM) by using T-43 (Oryza sativa L.)as material. The sowing date was the primary factor and sowing was conducted at soil temperature of 12℃(T1),15℃(T2),18℃(T3). The subplot was soil acidifiers: ammonium sulfate with nitrification inhibitor (AS), phosphoric acid (PP), citric acid (CA), and contrast (CK). The results showed that acidifier application significantly reduced soil pH value and increased soil iron availability with AS being the most effective one. Compared with CK the active iron concentration in rice leaf increased by 14.4%, 11.3% and 9.2%, respectively under As, PP and CA application. Acidifier application also improved root activity at seedling stage, root length, root surface area, and root volume, especially AS. Late sowing improved root activity of rice at seedling stage, increased root length, root surface area, and root volume. The active iron concentration in rice leaf under DIPM increased by 11.2% and SPAD value was significantly improved. In conclusion, application of acidifier helps overcome rice leaf chlorisis under DIPM, as well as delayed sowing date,with As showing the best effect.