Abstract Constitutive domains of repressive heterochromatin are maintained within the fission yeast genome through self-reinforcing mechanisms involving histone methylation and small RNAs. Non-coding RNAs generated from heterochromatic regions are processed into small RNAs by the RNA interference pathway, and are subject to silencing through both transcriptional and post-transcriptional mechanisms. While the pathways involved in maintenance of the repressive heterochromatin state are reasonably well understood, less is known about the requirements for its establishment. Here, we describe a novel role for the post-transcriptional regulatory factor Mkt1 in establishment of heterochromatin at pericentromeres in fission yeast. Loss of Mkt1 does not affect maintenance of existing heterochromatin, but does affect its recovery following depletion, as well as de novo establishment of heterochromatin on a mini-chromosome. Pathway dissection revealed that Mkt1 is required for RNAi-mediated post-transcriptional silencing, downstream of small RNA production. Mkt1 physically associates with pericentromeric transcripts, and is additionally required for maintenance of silencing and heterochromatin at centromeres when transcriptional silencing is impaired. Our findings provide new insight into the mechanism of RNAi-mediated post-transcriptional silencing in fission yeast, and unveil an important role for post-transcriptional silencing in establishment of heterochromatin that is dispensable when full transcriptional silencing is imposed.