Methylmercury (MeHg), a potent neurotoxin, easily moves from the soil into rice plants and subsequently accumulates within the grains. Although biochar can reduce MeHg accumulation in rice grains, the precise mechanism underlying biochar-mediated responses to mercury (Hg) stress, specifically regarding MeHg accumulation in rice, remains poorly understood. In the current study, we employed a 4% biochar amendment to remediate Hg-contaminated paddy soil, elucidate the impacts of biochar on MeHg accumulation through a comprehensive analysis involving Hg isotopic fractionation and transcriptomic analyses. The results demonstrated that biochar effectively lowered the levels of MeHg in paddy soils by decreasing bioavailable Hg and microbial Hg methylation. Furthermore, biochar reduced the uptake and translocation of MeHg in rice plants, ultimately leading to a reduction MeHg accumulation in rice grains. During the process of total mercury (THg) uptake, biochar induced a more pronounced negative isotope fractionation magnitude, whereas the effect was less pronounced during the upward transport of THg. Conversely, biochar caused a more pronounced positive isotope fractionation magnitude during the upward transport of MeHg. Transcriptomics analyses revealed that biochar altered the expression levels of genes associated with the metabolism of cysteine, glutathione, and metallothionein, cell wall biogenesis, and transport, which possibly enhance the sequestration of MeHg in rice roots. These findings provide novel insights into the effects of biochar application on Hg transformation and transport, highlighting its role in mitigating MeHg accumulation in rice.