Bitter gourd fruit are susceptible to chilling injury when stored at 4 °C. In this study, bitter gourds were stored at 20 °C to simulate normal ripening and senescence processes, and at 4 °C to induce chilling injury. Transcriptomic, metabolomic, and proteomic analyses were employed to investigate the molecular mechanisms of chilling injury development during low-temperature storage. The results showed that low-temperature storage induced the expression of genes related to proline metabolism (delta-1-pyrroline-5-carboxylate synthase-like, ornithine--oxo-acid transaminase, and proline dehydrogenase2, mitochondrial-like), antioxidants (glutathione peroxidase 2, superoxide dismutase, and L-ascorbate peroxidase 6), and membrane lipid metabolism. Additionally, low-temperature storage affected the ICE-CBF-COR pathway (calcium-binding protein CML15 and transcription factor ICE1). Correspondingly, free fatty acids (9,12,13-trihydroxy-10,15-octadecadienoic acid, 9-hydroperoxy-10E,12,15Z-octadecadienoic acid, γ-linolenic acid, α-linolenic acid*) and phospholipid metabolites LysoPC (18:4, 20:3, 19:1) and LysoPE (20:3, 20:3*, 20:2*) also accumulated during storage. Low-temperature storage increased POD and GPX activities. Overall, chilling injury in bitter gourd was associated with the induction of genes involved in proline metabolism, antioxidant activity, membrane lipid metabolism, cold perception mechanisms, and plant hormone signal transduction.