Abstract Background Rice blast caused by Magnaporthe oryzae is one of the most destructive diseases of rice. An increasing number of microRNAs (miRNAs) have been reported to fine-tune rice immunity against M. oryzae and coordinate with growth and development. Results Here, we showed that rice microRNA159a (Osa-miR159a) played a positive role in rice resistance to M. oryzae. The expression of Osa-miR159a was suppressed in a susceptible accession at 12, 24, and 48 h post-inoculation (hpi); it was upregulated in a resistant accession of M. oryzae at 24 hpi. The transgenic rice lines overexpressing Osa-miR159a were highly resistant to M. oryzae. In contrast, the transgenic lines expressing a short tandem target mimic (STTM) to block Osa-miR159a showed enhanced susceptibility. Knockout mutations of the target genes of Osa-miR159a, including OsGAMYB, OsGAMYBL, and OsZF, led to resistance to M. oryzae. Alteration of the expression of Osa-miR159a impacted yield traits including pollen and grain development. Conclusions Our results indicated that Osa-miR159a positively regulated rice immunity against M. oryzae by downregulating its target genes. Proper expression of Osa-miR159a was critical for coordinating rice blast resistance with grain development.