Biochar can effectively alleviate the Al phytotoxicity in acidic soils due to its alkaline nature. However, the longevity of this alleviation effect of biochar under re-acidification conditions is still unclear. In the present study, the maize root growth responding to the simulated re-acidification of two acidic soils amended by peanut straw biochar or Ca(OH)2 was investigated to evaluate the long-term effect of biochar on alleviating Al toxicity in acidic soils. Compared with Ca(OH)2 amendment, the application of biochar significantly retarded Al toxicity to plant during soil re-acidification. When 4.0 mM HNO3 was added, the maize seedling root elongation in an Oxisol with biochar was 99% higher than that in the Oxisol with Ca(OH)2. Also, the Evans blue uptake and Al content in the root tip in the biochar treatment were 60% and 51% lower than those in the Ca(OH)2 treatment. The retarding effect was mainly attributed to the slow decrease in soil pH during acidification and the release of dissolved organic carbon (DOC) in the soils amended by biochar. The slower decrease in soil pH resulting from the increased pH buffering capacity after biochar application inhibited the increase of soluble and exchangeable Al during re-acidification. The increased DOC after biochar application decreased the toxic soluble Al speciation at the same pH value and total Al concentration in soil solution. Therefore, given the re-acidification of soils, biochar presented a longer-term effect on alleviating Al toxicity of acidic soil than liming. Display omitted •Biochar retarded the Al toxicity to plant during soil re-acidification.•Biochar decreased the concentration of soil active Al during soil re-acidification.•Biochar altered the distribution of Al speciation in soil solution.•Biochar presented a long lasting effect on alleviating Al toxicity in acidic soils.