A reduction in the strong immunosuppressive activity of FK506 (1) is essential for developing this compound as an antifungal agent. Seven new FK506 analogues modified at both the FK506-binding protein 12- and the calcineurin-binding regions were biosynthesized. 9-DeoxoFK520 (7) exhibited a >900-fold reduction in the in vitro immunosuppressive activity but maintained significant antifungal activity, indicating that the C-9 and C-21 positions are critical for separation of immunosuppressive and antifungal activities. 7 exhibited robust synergistic antifungal activity with fluconazole. FK506 (1) is a 23-membered macrolide produced by several Streptomyces species and is used as an immunosuppressive drug to prevent the rejection of transplanted organs. FK506 has also exhibited antifungal, neuroprotective, and neuroregenerative activities. In humans, FK506 binds to FK506-binding protein (FKBP) 12, and the resulting FKBP12-FK506 complex interacts with a Ca2+-calmodulin-dependent phosphatase, calcineurin (CaN). Inactivation of CaN by forming the FKBP12-FK506-CaN ternary complex prevents the activation of nuclear factor of activated T cells (NF-AT), inhibiting the production of interleukin-2 and subsequent T-cell proliferation. This CaN signaling pathway also plays a critical role in the growth and pathogenesis of major fungal pathogens such as Cryptococcus neoformans, Candida albicans, and Aspergillus fumigatus. Therefore, the synthesis of FK506 analogues that can discriminate human FKBP12/CaN from its fungal counterparts may separate antifungal activity from the immunosuppressive activity, thereby allowing the development of a novel antifungal agent.