TRAIL is a death receptor ligand that induces cell death preferentially in tumor cells. Recombinant soluble TRAIL, however, performs poorly as an anti-cancer therapeutic because oligomerization is required for potent biological activity. We previously generated a diabody format of tumor-targeted TRAIL termed Db(αEGFR-sc)TRAIL, comprising single-stranded TRAIL molecules (scTRAIL) and the variable domains of a humanized variant of the EGFR blocking antibody Cetuximab. Here we define the bioactivity of Db(αEGFR)-scTRAIL with regard to both EGFR inhibition and TRAIL receptor activation in 3D cultures of Caco-2 colorectal cancer cells, which express wild-type K-Ras. Compared with conventional 2D cultures, Caco-2 cells displayed strongly enhanced sensitivity toward Db(αEGFR)-scTRAIL in these 3D cultures. We show that the antibody moiety of Db(αEGFR-sc)TRAIL not only efficiently competed with ligand-induced EGFR function, but also determined the apoptotic response by specifically directing Db(αEGFR)-scTRAIL to EGFR-positive cells. To address how aberrantly activated K-Ras, which leads to Cetuximab resistance, affects Db(αEGFR-sc)TRAIL sensitivity, we generated stable Caco-2tet cells inducibly expressing oncogenic K-Ras(G12V). In the presence of doxycycline, these cells showed increased resistance to Db(αEGFR-sc)TRAIL, associated with the elevated expression of the anti-apoptotic proteins cIAP2, Bcl-xL and FlipS. Co-treatment of cells with the Smac mimetic SM83 restored the Db(αEGFR)-scTRAIL-induced apoptotic response. Importantly, this synergy between Db(αEGFR)-scTRAIL and SM83 also translated to 3D cultures of oncogenic K-Ras expressing HCT-116 and LoVo colorectal cancer cells. Our findings thus support the notion that Db(αEGFR)-scTRAIL therapy in combination with apoptosis-sensitizing agents may be promising for the treatment of EGFR-positive colorectal cancers, independently of their KRAS status.