In plants, microRNA (miRNA) functions in the post-transcriptional repression of target mRNAs are well-explored, but the mechanisms regulating the accumulation of miRNAs remain poorly understood. Here, we report that distinct mechanisms regulate accumulation of a monocot-specific miRNA, rice (Oryza sativa) miR528. At the transcriptional level, miR528 accumulated in older plants and exhibited diurnal rhythms; at the post-transcriptional level, aging modulated miR528 levels by enhancing pri-miR528 alternative splicing. In addition, miR528 promoted flowering under long-day conditions by targeting RED AND FAR-RED INSENSITIVE 2 (OsRFI2). Moreover, natural variation in the MIR528 promoter region caused differences in miR528 expression among rice varieties, potentially due to differences in the binding strength of the transcription factor OsSPL9. Together, our findings show evidence for fine-tuning of miR528 levels in rice and provide insight into the mechanisms that regulate MIRNA expression in plants.