This study investigated the efficacy and tolerability of ZD9331 as second- or third-line treatment for patients with advanced colorectal cancer (aCRC). One hundred patients were recruited to the study: 45 in group 1 (failed first-line 5-FU-based regimen) and 55 in group 2 (failed first-line 5-FU-based regimen and second-line irinotecan). Patients received ZD9331 as a 30-minute intravenous infusion on days 1 and 8 of a 3-week cycle, and treatment continued until disease progression (PD) or withdrawal. After a median of 4 cycles of treatment, there were no objective responses in group 1 (N = 37), 25 (67.6%) patients had a best overall response of stable disease (SD), and 12 (32.4%) had PD. After a median of 3 cycles of treatment, there were 2 (4.5%) partial responses in group 2 (N = 44), 21 (47.7%) patients had a best overall response of SD, 20 (45.4%) had PD, and 1 (2.3%) had clinical progression. At data cut-off, 59.5% and 77.3% of patients in groups 1 and 2, respectively, had PD. The main adverse events were neutropenia (69%), fatigue (53%), nausea (46%), and diarrhea (40%), and most (72.3%) were grade I/II. ZD9331 demonstrated minimal antitumor activity, and manageable toxicity, in the second- or third-line treatment of aCRC.