Morphogenesis of many protozoans depends on a polarized establishment of cytoskeletal structures. In malaria‐causing parasites, this can be observed when a round zygote develops into an elongated motile ookinete within the mosquito stomach. This morphogenesis is mediated by the pellicle cytoskeletal structures, including the inner membrane complex (IMC) and the underlying subpellicular microtubules (SPMs). How the parasite maintains the IMC‐SPM connection and establishes a dome‐like structure of SPM to support cell elongation is unclear. Here, we show that palmitoylation of N‐terminal cysteines of two IMC proteins (ISP1/ISP3) regulates the IMC localization of ISP1/ISP3 and zygote‐to‐ookinete differentiation. Palmitoylation of ISP1/ISP3 is catalyzed by an IMC‐residing palmitoyl‐S‐acyl‐transferase (PAT) DHHC2. Surprisingly, DHHC2 undergoes self‐palmitoylation at C‐terminal cysteines via its PAT activity, which controls DHHC2 localization in IMC after zygote formation. IMC‐anchored ISP1 and ISP3 interact with microtubule component β‐tubulin, serving as tethers to maintain the proper structure of SPM during zygote elongation. This study identifies the first PAT–substrate pair in malaria parasites and uncovers a protein palmitoylation cascade regulating microtubule cytoskeleton. Synopsis: In Plasmodium, zygote to ookinete morphogenesis depends on a polarized establishment of pellicle cytoskeleton. Interaction between palmitoylated ISP1/ISP3 and β‐tubulin plays a bridging role in connecting the inner membrane complex and underlying subpellicular microtubules. Polarized localization of inner membrane complex proteins ISP1/ISP3 controls zygote‐to‐ookinete morphogenesis.Palmitoylation is critical for IMC localization and function of ISP1/ISP3.Self‐palmitoylated DHHC2 translocates to IMC and palmitoylates ISP1/ISP3.ISP1/ISP3 maintains subpellicular microtubule network organization by interacting with β‐tubulin. [ABSTRACT FROM AUTHOR]