Auxin is important in regulating bamboo growth and development. Naturally grown moso bamboo shoots were treated with auxin, and changes in endogenous hormones were determined by a selective response/multi-response monitoring–targeted metabolomics approach. Gibberellin and cytokinin were significantly upregulated, while auxin inhibitor treatment also resulted in endogenous hormone disruption. Transcriptome sequencing using Oxford Nanopore Technology revealed that the regulation of internode length by auxin is mainly through three pathways at the transcriptional, translational and post-translational levels. The pathways are phytohormone signaling, amino acid synthesis and protein processing, and energy metabolism. The transcription factors that play major regulatory roles include ethylene response factor, WRKY and MYB transcription factor, NAC structural domain protein and basic leucine zipper protein. A gene regulatory network was constructed and screened, and we cloned the core regulatory gene— PheAUX/IAA34. We found that there may be crosstalk between auxin signaling and jasmonic acid signaling, and verified that auxin can co-regulate growth with plant hormones by transgenic and yeast two-hybrid experiments. The results demonstrated that auxin-mediated height regulation in bamboo mainly regulates internode length rather than node numbers. For single internodes, auxin mainly regulates internode elongation by promoting cell elongation. High-quality bamboo internode materials for production can be obtained by auxin treatment, which can provide a reference for bamboo plant height regulation research.