<b><i>Background:</i></b> The common cytotoxic mechanisms that underpin chemoefficacy and toxicity have hampered efforts to deliver effective supportive care interventions, particularly for gastrointestinal (GI) toxicity. Matrix metalloproteinases (MMPs) have been implicated in both tumor growth and GI toxicity, and as such MMP inhibitors present as a novel therapeutic avenue to simultaneously enhance treatment efficacy and reduce toxicity. <b><i>Objectives:</i></b> The aim of this study was to determine the efficacy of an MMP-9/12 inhibitor, AZD3342, on tumor growth and GI toxicity in a rat model. <b><i>Methods:</i></b> Female tumor-bearing Dark Agouti rats (<i>n</i> = 90) were divided into 4 groups: vehicle control; methotrexate (MTX); AZD3342, and MTX + AZD3342. Tumors were measured daily (for 5 days) using digital calipers. GI toxicity was assessed using well-established clinical markers (diarrhea/weight loss), histopathological analysis, and functional assessment of intestinal barrier permeability. <b><i>Results:</i></b> AZD3342 delayed the onset of severe diarrhea by 1 day (vs. MTX) but was unable to improve the overall severity of diarrhea. No changes were detected in tissue morphology or intestinal barrier function. AZD3342 alone suppressed tumor growth (<i>p</i> = 0.003 vs. vehicle) but did not enhance the efficacy of MTX. <b><i>Conclusions:</i></b> This study showed partial efficacy of AZD3342 in reducing tumor growth and delaying the onset of severe diarrhea caused by MTX in rats. We suggest further studies be undertaken targeting appropriate scheduling of AZD3342 as well as investigating different cytotoxic therapies that strongly activate MMP signaling.