Boron (B) deficiency affects the expressions of genes involved in major physiological processes. However, signal transduction pathway through which plants are able to sense and transmit B-deprivation signal to the nucleus is unknown. The aim of this work was to research in Arabidopsis thaliana roots whether the short-term B deficiency affects cytosolic Ca2+ levels ([Ca2+]cyt) as well as expression of genes involved in Ca2+ signaling. To visualize in vivo changes in root [Ca2+]cyt, Arabidopsis seedlings expressing Yellow Cameleon (YC) 3.6 were grown in a nutrient solution supplemented with 2 μM B and then transferred to a B-free medium for 24 h. Root [Ca2+]cyt was clearly higher in B-deficient seedlings upon 6 and 24 h of B treatments when compared to controls. Transcriptome analyses showed that transcript levels of Ca2+ signaling-related genes were affected by B deprivation. Interestingly, Ca2+ channel (CNGC19, cyclic nucleotide-gated ion channel) gene was strongly upregulated as early as 6 h after B deficiency. Expression levels of Ca2+ transporter (ACA, autoinhibited Ca2+-ATPase; CAX, cation exchanger) genes increased when seedlings were subjected to B deficiency. Gene expressions of calmodulin-like proteins (CMLs) and Ca2+-dependent protein kinases (CPKs) were also overexpressed upon exposure to B starvation. Our results suggest that B deficiency causes early responses in the expression of CNGC19 Ca2+-influx channel, ACA- and CAX-efflux, and Ca2+ sensor genes to regulate Ca2+ homeostasis. It is the first time that changes in the levels of in vivo cytosolic Ca2+ and expression of Ca2+ channel/transporter genes are related with short-term B deficiency in Arabidopsis roots. © 2013 Elsevier Masson SAS.