High mobility group box 1 (HMGB1) is a critical pro-inflammatory damage-associated molecular pattern (DAMP), and ribosomal protein L9 (RPL9) has been identified as a potential “regulatory DAMP” that can suppress HMGB1’s activity. However, it is unclear how pro-inflammatory DAMP signaling is initiated when these opposing molecules are released together. We hypothesized that the release kinetics of HMGB1 and RPL9 are differentially regulated depending on the cell type. To test this, we compared the release of HMGB1 and RPL9 from human macrophage (THP-1), liver (HepG2), and endothelial (EA.hy926) cell lines following stimulation with lipopolysaccharide and nigericin. In THP-1 macrophages, classical pyroptosis induced a rapid, sequential release of HMGB1 followed by RPL9. In contrast, HepG2 cells showed slower, apoptosis-like cell death, and RPL9 was released several hours before HMGB1. EA.hy926 cells were highly resistant to the stimulus. Notably, RPL9 release was closely associated with phosphatidylserine externalization in both THP-1 and HepG2 cells, regardless of their primary cell death pathway. Our findings demonstrate that the balance between pro-inflammatory and regulatory DAMPs is governed by a complex, cell-type-specific temporal control system intrinsically linked to the mode of cell death. Furthermore, we propose the existence of a novel, selective release pathway for RPL9. A deeper understanding of this time-dependent regulation may contribute to the development of new therapeutic strategies for inflammatory diseases.