Heap bioleaching is presently the most successful technology for copper extraction from low-grade sulfide ores. Metabolic behaviors and adaptation mechanisms of microbial communities to this system remain unknown. A combination of molecular approaches has been used to determine the key microorganisms in the Escondida Sulphide Heap. This work reports the global gene expression in industrial bioleaching samples by using a specific DNA microarray of Leptospirillum ferrooxidans to better understand the metabolic functions displayed by Leptospirillum spp. iron oxidizers as mineral leaching proceeds. Quantitative real-time PCR analysis showed low cell numbers of Leptospirillum species during the first part of the irrigation period, however, it became the most abundant and active in the second part of the cycle. Transcriptome analysis by DNA microarray could provide specifics transcription patterns in the different stages of the industrial process. We studied the gene expression from bioleaching heap samples with variable Leptospirillum cell numbers, operation times and with different chemical and physical conditions. The results showed induction of genes involved in energetic metabolism, electron transport, ribosomal proteins, DNA repair and response to heavy metals when Leptospirillum species were most abundant and active. We suggest that up- and down-regulation of specifics genes could be used as indicators of physiological responses to operational conditions in the bioleaching systems.