The rubber tree currently serves as the sole source of natural rubber (NR). However, its limited cultivation range and the increasing global NR demand necessitate the development of an alternative crop for NR production. This study reports that Lactuca serriola can produce high-quality NR suitable for industrial rubber demand. The rubber molecular weight of L. serriola exceeds 750 kg/mol, with NR production occurring throughout the entire plant. Furthermore, treatments with ethylene, methyl jasmonate (MeJA), and salicylic acid (SA) significantly increased rubber content in L. serriola. Transcriptome analysis revealed that ethylene and MeJA treatments affected gene expression associated with isopentenyl pyrophosphate (IPP) synthesis, while ethylene and SA treatments influenced gene expression involved in sucrose transportation and metabolism. Through Pearson correlation coefficient (PCC) analysis and virus-induced gene silencing, several transcription factors and LsCPTs/LsCPTL were identified as key regulators of rubber synthesis in L. serriola. Yeast two-hybrid and co-expression assays suggested that LsCPTL anchors LsCPT1 and LsCPT2 to the endoplasmic reticulum, forming a protein complex that regulates rubber synthesis. This study provides a preliminary analysis of the mechanism by which plant hormones regulate rubber synthesis in L. serriola, revealing its significant potential as an alternative to the rubber tree for NR production.