Background Cachexia is a significant problem in patients with cancer. The effect of cancer on interstitial cells of Cajal (ICC) and neurons of the gastrointestinal tract have not been studied previously. While supplementation with L-glutamine 2% may have beneficial effects in cancer-related cachexia, and be protective of ICC in models of oxidative stress such as diabetes, its effects on ICC in cancer have also not been studied. Methods Twenty-eight male Wistar rats were divided into four groups: control (C), control supplemented with L-glutamine (CG), Walker 256 tumor (WT), and Walker 256 tumor supplemented with L-glutamine (WTG). Rats were implanted with tumor cells or injected with saline in the right flank. After 14 days, the jejunal were collected and processed for immunohistochemical techniques including whole mounts and cryosections and Western blot analysis. Key Results Tumor-bearing rats demonstrate reduced numbers of ICC-MY and ICC-DMP and yet increased Ano1 protein expression and enhanced ICC networks. In addition, there is more nNOS protein expressed in tumor-bearing rats compared to controls. L-glutamine treatment had a variety of effects on ICC that may be related to disease state and the interaction of ICC and nNOS neurons. Regardless, L-glutamine reduced the size of tumors and also tumor-induced cachexia that was not due to altered food intake. Conclusions & Inferences There are significant effects on ICC in the Walker 256 tumor model. While, supplementation with L-glutamine has differential and complex effects of ICC, it reduces tumor size and tumor-associated cachexia, which supports its beneficial therapeutic role in cancer.