Instant and accurate evaluation of drug resistance in tumors before and during chemotherapy is important for patients with advanced colon cancer and is beneficial for prolonging their progression-free survival time. Here, the possible biomarkers that reflect the drug resistance of colon cancer were investigated using proton magnetic resonance spectroscopy (1H-MRS) in vivo. SW480[5-fluorouracil(5-FU)-responsive] and SW480/5-FU (5-FU-resistant) xenograft models were generated and subjected to in vivo 1H-MRS examinations when the maximum tumor diameter reached 1–1.5 cm. The areas under the peaks for metabolites, including choline (Cho), lactate (Lac), glutamine/glutamate (Glx), and myo-inositol (Ins)/creatine (Cr) in the tumors, were analyzed between two groups. The resistance-related protein expression, cell morphology, necrosis, apoptosis, and cell survival of these tumor specimens were assessed. The content for tCho, Lac, Glx, and Ins/Cr in the tumors of the SW480 group was significantly lower than that of the SW480/5-FU group (P < 0.05). While there was no significant difference in the degree of necrosis and apoptosis rate of tumor cells between the two groups (P > 0.05), the tumor cells of the SW480/5-FU showed a higher cell density and larger nuclei. The expression levels of resistance-related proteins (P-gp, MPR1, PKC) in the SW480 group were lower than those in the SW480/5-FU group (P < 0.01). The survival rate of 5-FU-resistant colon cancer cells was significantly higher than that of 5-FU-responsive ones at 5-FU concentrations greater than 2.5 μg/mL (P < 0.05). These results suggest that alterations in tCho, Lac, Glx1, Glx2, and Ins/Cr detected by 1H-MRS may be used for monitoring tumor resistance to 5-FU in vivo.