We explored whether the combination of lurbinectedin (PM01183) with the antimetabolite gemcitabine may result in synergistic antitumor effect in pancreatic adenocarcinoma (PDA) models. We also studied the contribution of lurbinectedin to this synergism. This drug presents a dual pharmacological effect that contributes to its in vivo antitumor activity: (i) specific binding to DNA minor groove inhibiting active transcription and DNA repair; and (ii) specific depletion of tumor-associated macrophages (TAMs). We evaluated the in vivo antitumor activity of lurbinectedin, gemcitabine (as single agents) and its combination in SW-1990 and MIA PaCa-2 cell-line xenografts and in patient-derived PDA models (AVATAR). Lurbinectedin-gemcitabine combination induced a synergistic effect on both, MIA PaCa-2 (CI=0.66) and SW1990 (CI=0.80) tumor xenografts. It also induced complete tumor remissions in 4 of 6 patient-derived PDA xenografts. This synergism was associated with enhanced DNA damage (anti-γ-H2AX), cell cycle blockage, caspase-3 activation and apoptosis. In addition to the enhanced DNA damage, which is a consequence of the interaction of the two drugs with the DNA, lurbinectedin induced TAMs depletion leading to CDA down-regulation in PDA tumors. This effect could, in turn, induce an increase of gemcitabine-mediated DNA damage that was especially relevant in high-density TAMs tumors. These results show that lurbinectedin can be used to develop “molecularly-targeted” combination strategies.