The incidence of diabetes after organ transplantation (NODAT) has increased, in part due to calcineurin inhibitors, particularly tacrolimus (TAC). Voclosporin (VCS) is a next generation calcineurin inhibitor that causes less NODAT in clinical trials, but its effects on human beta cells are unknown. We compared the effects of TAC and VCS on the dynamics of insulin secretory function, the programmed cell death rate and the transcriptomic profile of isolated human islets. We studied 2 clinically relevant doses of TAC (10 ng/ml, 30 ng/ml) and VCS (20 ng/ml, 60 ng/ml), meant to approximate the trough and peak concentrations of each drug. We found that the TAC, but not VCS, caused a significant impairment of both 15 mM glucose-stimulated insulin secretion and 30 mM KCl-stimulated insulin secretion, pointing to a possible molecular defect in the distal stages of exocytosis after Ca2+ entry via voltage-gated Ca2+ channels. Both TAC and VCS inhibited insulin secretion at high concentrations. No effects on insulin contents were identified. RNA sequencing showed that TAC, and to a lesser extent VCS, decreased the expression of genes that regulate exocytosis, including synaptotagmins (SYT16, SYT5), ER-to-Golgi traffic (PITPNM1) and hormone processing (PAM). Pathway analysis showed a significant enrichment of Gene Ontology terms, including ‘syntaxin binding.’ No significant deleterious effects on cell survival were observed with either drug in this model. Collectively, these data suggest that calcineurin inhibitors can directly inhibit insulin exocytosis. These data also support prior clinical evidence demonstrating the favourable glucose homeostasis profile of VCS when compared to TAC.