Abstract Drpl is a dynamin GTPase important for mitochondrial and peroxisomal division. Drp1 oligomerization and mitochondrial recruitment are regulated by multiple factors, including interaction with mitochondrial receptors such as Mff, MiD49, MiD51 and Fis. In addition, both endoplasmic reticulum (ER) and actin filaments play positive roles in mitochondrial division, but mechanisms for their roles are poorly defined. Here, we find that a population of Drp1 oligomers is ER-associated in mammalian cells, and is distinct from mitochondrial or peroxisomal Drp1 populations. Sub-populations of Mff and Fis1, which are tail-anchored proteins, also localize to ER. Drp1 oligomers assemble on ER, from which they can transfer to mitochondria. Suppression of Mff or inhibition of actin polymerization through the formin INF2 significantly reduces all Drp1 oligomer populations (mitochondrial, peroxisomal, ER-bound) and mitochondrial division, while Mff targeting to ER has a stimulatory effect on division. Our results suggest that ER can function as a platform for Drp1 oligomerization, and that ER-associated Drp1 contributes to mitochondrial division. Summary Assembly of the dynamin GTPase Drp1 into constriction-competent oligomers is a key event in mitochondrial division. Here, Ji et al show that Drp1 oligomerization can occur on endoplasmic reticulum through an ER-bound population of the tail-anchored protein Mff. Abbreviations used in this paper: Drp1, dynamin-related protein 1; Fis1, mitochondrial fission 1 protein; INF2, inverted formin 2; KD, siRNA-mediated knock down; KI, CRISPR-mediated knock in; KO, CRISPR-mediated knock out; LatA, Latrunculin A; MDV, mitochondrially-derived vesicle; Mff, mitochondrial fission factor; MiD49 and MiD51, mitochondrial dynamics protein of 49 and 51 kDa; OMM, outer mitochondrial membrane; TA, tail-anchored.