Plants play an important role in the global sulphur cycle because they assimilate sulphur from the environment and build it into methionine and cysteine. Several genes of the sulphur assimilation pathway are regulated by microRNA-395 (miR395) that is itself induced by a low-sulphur (−S) environment. Here, we show that the six Arabidopsis miR395 loci are induced differently. We find that MIR395 loci are expressed in the vascular system of roots and leaves and root tips. Induction of miR395 by a −S environment in both roots and leaves suggests that translocation of miR395 from leaves to roots through the phloem is not necessary for plants growing on −S soil/medium. We also demonstrate that induction of miR395 is controlled by SLIM1, a key transcription factor in the sulphur assimilation pathway. Unexpectedly, the mRNA level of a miR395 target gene, SULTR2;1, strongly increases during miR395 induction in roots. We show that the spatial expression pattern of MIR395 transcripts in the vascular system does not appear to overlap with the expression pattern previously reported for SULTR2;1 mRNA. These results illustrate that negative temporal correlation between the expression level of a miRNA and its target gene in a complex tissue cannot be a requirement for target gene validation.