AIM: To investigate the role of exosomal miR-29b and Ca2+ in regulating the function of human lens epithelial cells (HLECs). METHODS: Exosomes were isolated from human aqueous humour (AH) by ultracentrifugation, and visualized by nanoparticle tracking and transmission electron microscopy. Exosomal miRNA sequencing was performed to identify differentially expressed miRNAs between diabetes with cataracts (DMC) group and age-related cataracts (ARC) group. TargetScan was used to predict potential target of certain miRNA. The expression of CACNA1C mRNA was determined by quantitative real-time polymerase chain reaction and CACNA1C protein was determined by Western blotting. Concentration of Ca2+ in human AH and the culture supernatant of cells were detected by the calcium assay kit. Cell counting kit-8 was used to determine cell viability. RESULTS: Exosomes were isolated from human AH, which had a typical cup-shaped phenotype and a particle size distribution in accordance with micro extracellular vesicles. Exosomal miRNA sequencing revealed that miR-29b was significantly downregulated in DMC group compared with ARC. Ca2+ concentration of human AH in DMC was higher than that in ARC. The culture supernatant of cells transfected with miR-29b inhibitors had a higher concentration of Ca2+ than that transfected with miR-29b mimics. miR-29b reduced the viability of HLECs by upregulating CACNA1C expression. CONCLUSION: Exosomes isolated from human AH contains abundant miRNAs. A significantly expressed miRNA, miR-29b, can affect the concentration of Ca2+ and regulate HLEC processes by upregulating CACNA1C.