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Naphthylacetic acid (NAA) was used to increase the tuberous root yield of <i>Rehmannia glutinosa</i>, but the differences between its NAA-treated and control tuberous roots (NT and CG) and the <i>r</i>egulatory mechanism of NAA effect remain unclear. In order to investigate them, NTs and CGs were used as materials, and both yield-related indices were measured; the metabolomics and transcriptomics were used to capture differentially accumulated metabolites (DAM) and to validate them via mining differentially expressed genes (DEGs), respectively. The effects of NAA treatment: increased NT mass per plant by 21.14%, through increasing the number of roots and increasing the mean root diameter; increased catalpol content by 1.2234% (<i>p</i> < 0.05); up-regulated 11DAMs and 596DEGs; and down-regulated 18 DAMs and 517DEGs. In particular, we discovered that NAA regulated its DAMs and biomass via 10 common metabolic pathways, and that the number of NAA-down-regulated DAMs was more than that of NAA-up-regulated DAMs in its tuberous root. Furthermore, HPLC validated the changes of several DAMs and 15 DEGs (<i>4CL</i>, <i>ARF</i>, <i>CCoAOMT</i>, <i>A</i><i>RGOS</i>, etc.) associated with the yield increase and DAMs were verified by RT-qPCR. This study provided some valuable resources, such as tuberous root indices, key genes, and DAMs of <i>Rehmannia glutinosa</i> in response to NAA for distinguishing the CGs from NTs, and novel insights into the <b>r</b>egulatory mechanism of NAA effects on both at the transcriptomic and metabolomic levels, so it will lay a theoretical foundation for NAA-regulated plant yield and quality, and provide references for prohibiting the uses of NAA as a swelling agent in medicinal tuber plants in China.