Huntington’s disease is a neurodegenerative disorder caused by an expansion of CAGs repeats and characterized by alterations in mitochondrial functions. Although changes in Ca2+ handling have been suggested, the mechanisms involved are not completely understood. The aim of this study was to investigate the possible alterations in Ca2+ handling capacity and the relationship with mitochondrial dysfunction evaluated by NAD(P)H fluorescence, reactive oxygen species levels, mitochondrial membrane potential (ΔΨm) measurements and respiration in whole brain slices from R6/1 mice of different ages, evaluated in situ by real-time real-space microscopy. We show that the cortex and striatum of the 9-month-old R6/1 transgenic mice present a significant sustained increase in cytosolic Ca2+ induced by glutamate (Glu). This difference in Glu response was partially reduced in R6/1 when in the absence of extracellular Ca2+, indicating that N-methyl-d-aspartate receptors participation in this response is more important in transgenic mice. In addition, Glu also lead to a decrease in NAD(P)H fluorescence, a loss in ΔΨm and a further increase in respiration, which may have evoked a decrease in mitochondrial Ca2+ () uptake capacity. Taken together, these results show that alterations in Ca2+ homeostasis in transgenic mice are associated with a decrease in uptake mechanism with a diminished Ca2+ handling ability that ultimately causes dysfunctions and worsening of the neurodegenerative and the disease processes.