New therapeutics and combination regimens have led to marked clinical improvements for the treatment of a subset of colorectal cancer (CRC). Immune checkpoint inhibitors have shown clinical efficacy in patients with mismatch repair-deficient or microsatellite instability-high (MSI-H) metastatic CRC (mCRC). However, patients with microsatellite-stable (MSS) or low levels of microsatellite instable (MSI-L) CRC have not benefited from these immune modulators, and the survival outcome remains poor for the majority of patients diagnosed with mCRC. In this report, we describe the discovery of a novel T cell-dependent bispecific antibody (TDB) targeting tumor-associated antigen LY6G6D for the treatment of CRC. Expression analyses reveal limited expression of LY6G6D in normal human tissues and non-CRC tumor types; meanwhile, significant LY6G6D upregulation in CRC is observed with high prevalence in MSS and MSI-L CRC subsets. The optimized anti-LY6G6D/CD3 TDB, which targets a membrane-proximal epitope of LY6G6D and binds to CD3 with high affinity, exhibits potent anti-tumor activity both in vitro and in vivo. In vitro functional assays show that LY6G6D-TDB-mediated T-cell activation and cytotoxicity are conditional and target-dependent. In mouse xenograft tumor models, LY6G6D-TDB demonstrates anti-tumor efficacy as a single agent against established CRC tumors, and enhanced efficacy can be achieved when LY6G6D-TDB is combined with PD-1 blockade. Our studies provide evidence for the therapeutic potential of anti-LY6G6D/CD3 TDB as an effective treatment option for patients with CRC.