Abstract Pregnenolone (P5) is a neurosteroid that improves memory and neurological recovery. It is also required for zebrafish embryonic development. However, its mode of action is unclear. Here I show that P5 promotes cell migration and microtubule polymerization by binding to a microtubule-plus-end-tracking cytoplasmic linker protein 1 (CLIP-170). CLIP-170 was captured from zebrafish embryonic extract by a P5 photoaffinity probe conjugated to diaminobenzophenone and identified by LC-MS/MS analysis. P5 interacted with CLIP-170 at its coiled-coil domain and changed it into an extended conformation. This increased CLIP-170 interaction with microtubules, dynactin subunit p150Glued, and LIS1; it also promoted CLIP-170-dependent microtubule polymerization. CLIP-170 was essential for P5 to promote microtubule abundance and zebrafish epiboly migration during embryogenesis; overexpression of the P5-binding region of CLIP-170 delayed this migration. P5 also sustained migration directionality of cultured mammalian cells. My results show that P5 regulates microtubule polymerization and cell migration via activating CLIP-170. Besides regulating microtubules, P5 also promoted resistance to the F-actin disrupting agent. P5 or CLIP-170 deficient embryos showed abnormal F-actin architectures and decreased endocytotic activity. P5 controlled interaction of CLIP-170 to the small GTPase Rho effector, mDia1. It suggests that P5 and CLIP-170 stabilize F-actin via mDia1. Together, P5 and CLIP-170 promote zebrafish embryonic movement by controlling both microtubule and F-actin.