The pleckstrin homology (PH) domain is a versatile fold that mediates a variety of protein–protein and protein–phosphatidylinositol lipid interactions. The Ran-binding protein 2 (RanBP2) contains four interspersed Ran GTPase-binding domains (RBD n = 1–4 ) with close structural homology to the PH domain of Bruton's tyrosine kinase. The RBD 2 , kinesin-binding domain (KBD) and RBD 3 comprise a tripartite domain (R 2 KR 3 ) of RanBP2 that causes the unfolding, microtubule binding and biphasic activation of kinesin-1, a crucial anterograde motor of mitochondrial motility. However, the interplay between Ran GTPase and R 2 KR 3 of RanBP2 in kinesin-1 activation and mitochondrial motility is elusive. We use structure–function, biochemical, kinetic and cell-based assays with time-lapse live-cell microscopy of over 260 000 mitochondrial-motility-related events to find mutually exclusive subdomains in RBD 2 and RBD 3 towards Ran GTPase binding, kinesin-1 activation and mitochondrial motility regulation. The RBD 2 and RBD 3 exhibit Ran-GTP-independent, subdomain and stereochemical-dependent discrimination on the biphasic kinetics of kinesin-1 activation or regulation of mitochondrial motility. Further, KBD alone and R 2 KR 3 stimulate and suppress, respectively, multiple biophysical parameters of mitochondrial motility. The regulation of the bidirectional transport of mitochondria by either KBD or R 2 KR 3 is highly coordinated, because their kinetic effects are accompanied always by changes in mitochondrial motile events of either transport polarity. These studies uncover novel roles in Ran GTPase-independent subdomains of RBD 2 and RBD 3 , and KBD of RanBP2, that confer antagonizing and multi-modal mechanisms of kinesin-1 activation and regulation of mitochondrial motility. These findings open new venues towards the pharmacological harnessing of cooperative and competitive mechanisms regulating kinesins, RanBP2 or mitochondrial motility in disparate human disorders.