Microtubule biogenesis initiates at various intracellular sites, including the centrosome, the Golgi apparatus, the nuclear envelope, and preexisting microtubules. Similarly, in the fission yeast Schizosaccharomyces pombe, interphase microtubules are nucleated at the spindle pole body (SPB), the nuclear envelope, and preexisting microtubules, depending on Mto1 activity. Despite the essential role of Mto1 in promoting microtubule nucleation, how distribution of Mto1 in different sites is regulated has remained elusive. Here, we show that the J-domain cochaperone Rsp1 interacts with Mto1 and specifies the localization of Mto1 to non-SPB nucleation sites. The absence of Rsp1 abolishes the localization of Mto1 to non-SPB nucleation sites, with concomitant enrichment of Mto1 to the SPB and the nuclear envelope. In contrast, Rsp1 overexpression impairs the localization of Mto1 to all microtubule organization sites. These findings delineate a previously uncharacterized mechanism in which Rsp1-Mto1 interaction orchestrates non-SPB microtubule formation.