Among cereal crops, rice is considered the most tolerant to aluminium (Al). However, variability among rice genotypes leads to remarkable differences in the degree of Al tolerance for distinct cultivars. A number of studies have demonstrated that rice plants achieve Al tolerance through an unknown mechanism that is independent of root tip Al exclusion. We have analysed expression changes of the rice ASR gene family as a function of Al treatment. The gene ASR5 was differentially regulated in the Al-tolerant rice ssp. Japonica cv. Nipponbare. However, ASR5 expression did not respond to Al exposure in Indica cv. Taim rice roots, which are highly Al sensitive. Transgenic plants carrying RNAi constructs that targeted the ASR genes were obtained, and increased Al susceptibility was observed in T1 plants. Embryogenic calli of transgenic rice carrying an ASR5-green fluorescent protein fusion revealed that ASR5 was localized in both the nucleus and cytoplasm. Using a proteomic approach to compare non-transformed and ASR-RNAi plants, a total of 41 proteins with contrasting expression patterns were identified. We suggest that the ASR5 protein acts as a transcription factor to regulate the expression of different genes that collectively protect rice cells from Al-induced stress responses.