The study investigates the role of tumor necrosis factor alpha (TNFα) in modulating pannexin 1 (PANX1) activation and ATP release to enhance P2RX7-mediated antitumor immune responses after chemotherapy in colorectal cancer (CRC). TNFα promotes PANX1 cleavage via a caspase 8/3-dependent pathway, enhancing cancer cell immunogenicity and leading to dendritic cell maturation and T-cell activation. Blockade of the ATP receptor P2RX7 by small molecules reduces the therapeutic efficacy of chemotherapy and decreases immune cell infiltration. Conversely, administration of an ATP mimic increases chemotherapy efficacy and immune cell infiltration. High PANX1 expression is positively correlated with DC and T-cell recruitment in the tumor microenvironment and is associated with favorable survival outcomes in CRC patients receiving adjuvant chemotherapy. Additionally, a loss-of-function P2RX7 mutation is linked to reduced CD8+ immune cell infiltration and poor survival outcomes. These findings highlight the importance of TNFα-mediated PANX1 cleavage in promoting ATP-P2RX7 signaling and enhancing chemotherapy-induced antitumor immunity.The study investigates the role of tumor necrosis factor alpha (TNFα) in modulating pannexin 1 (PANX1) activation and ATP release to enhance P2RX7-mediated antitumor immune responses after chemotherapy in colorectal cancer (CRC). TNFα promotes PANX1 cleavage via a caspase 8/3-dependent pathway, enhancing cancer cell immunogenicity and leading to dendritic cell maturation and T-cell activation. Blockade of the ATP receptor P2RX7 by small molecules reduces the therapeutic efficacy of chemotherapy and decreases immune cell infiltration. Conversely, administration of an ATP mimic increases chemotherapy efficacy and immune cell infiltration. High PANX1 expression is positively correlated with DC and T-cell recruitment in the tumor microenvironment and is associated with favorable survival outcomes in CRC patients receiving adjuvant chemotherapy. Additionally, a loss-of-function P2RX7 mutation is linked to reduced CD8+ immune cell infiltration and poor survival outcomes. These findings highlight the importance of TNFα-mediated PANX1 cleavage in promoting ATP-P2RX7 signaling and enhancing chemotherapy-induced antitumor immunity.