MicroRNAs (miRNAs) are endogenous small RNAs that can have large-scale regulatory effects on development and on stress responses in plants. The endemic rust fungus Cronartium quercuum f. sp. fusiforme causes fusiform rust disease in pines, resulting in the development of spindle-shaped galls (cankers) on branches or stems. This disease is the most destructive disease of pines in the southern USA. To test whether miRNAs play roles in fusiform rust gall development, we cloned and identified 26 miRNAs from stem xylem of loblolly pine (Pinus taeda), which belong to four conserved and seven loblolly pine-specific miRNA families. Forty-three targets for nine of these 11 families were experimentally validated in vivo. Sequence analysis suggested that the target cleavage site may be determined not only by the miRNA sequence but also by the target sequence. Members of three loblolly pine-specific miRNA families target a large number of non-protein coding transcripts, and one of these families could also initiate secondary phased production from its target of a putative trans-acting short interfering RNA (ta-siRNA). Expression of 10 of these 11 miRNA families was significantly repressed in the galled stem. PCR-based transcript quantification showed complex expression patterns of these miRNAs and their targets in the galled tissues and in tissues surrounding the gall. We further predict 82 plant disease-related transcripts that may also response to miRNA regulation in pine. These results reveal a new genetic basis for host-pathogen interactions in the development of fusiform rust gall.