CIGB-300 is a proapoptotic peptide-based drug that abrogates the CK2-mediated phosphorylation. This peptide has antineoplastic effect on lung cancer cells in vitro and in vivo. To understand the mechanisms involved on such anticancer activity, the NCI-H125 cell line proteomic profile after short-term incubation (45 min) with CIGB-300 was investigated. As determined by 2-DE or 2D-LC-MS/MS, 137 proteins changed their abundances more than 2-fold in response to the CIGB-300 treatment. The expression levels of proteins related to ribosome biogenesis, metastasis, cell survival and proliferation, apoptosis, and drug resistance were significantly modulated by the presence of CIGB-300. The protein translation process was the most affected (23% of the identified proteins). From the proteome analysis of the NCI-H125 cell line, novel potentialities for CIGB-300 as anticancer agent were evidenced.