Endogenous methyl jasmonate (MeJA) mediates abiotic and biotic stresses in plants. Exogenous MeJA application can stimulate and defend plant gene expression and induce plant chemical defense. The effects of foliar MeJA application on yield and 2-acetyl-1-pyrroline (2-AP) biosynthesis of fragrant rice are scarcely investigated. The pot experiment was conducted by spraying different concentrations of MeJA (0, 1, and 2 μM; denoted as CK, MeJA-1, and MeJA-2) at the initial heading stage of two fragrant rice cultivars, Meixiangzhan and Yuxiangyouzhan. The results showed that foliar MeJA application significantly increased the grain 2-AP content by 32.1% and 49.7%, respectively, following MeJA-1 and MeJA-2 treatments, and the two cultivars showed the highest 2-AP content upon MeJA-2 treatment. However, the grain yield was increased in MeJA-1 as compared with MeJA-2 treatment for all rice cultivars and no significant differences were observed in yield and yield-related traits compared with CK. The aroma was improved by foliar MeJA application which was strongly associated with the regulation of the precursors and enzymes involved in 2-AP biosynthesis. In particular, the contents of proline, pyrroline-5-carboxylic acid, and pyrroline at maturity, as well as the activities of proline dehydrogenase, ornithine aminotransferase, and pyrroline-5-carboxylic acid synthetase, were positively correlated with grain 2-AP content. On the other hand, foliar MeJA application improved the contents of soluble protein, chlorophyll a and b, and carotenoid, and increased the activity of antioxidant enzymes. Moreover, peroxidase activity and leaf chlorophyll contents were significantly positively correlated to 2-AP content following foliar MeJA application. Therefore, our results implied that foliar MeJA application increased aroma and influenced yield by regulating the physio-biochemistry characters and resistance, and suggested that the optimal concentration of MeJA for the best positive effect on the yield and aroma was 1 μM. However, further study is required to evaluate the metabolic level and molecular basis of the regulatory mechanism of foliar MeJA application on 2-AP in fragrant rice.