A smart contrast agent for magnetic resonance imaging (MRI) can be used to exploit an enzymatic activity specific to the tissue or disease state signified by converting an MRI-inactivated agent to an activated MRI agent. In this study, a β-galactopyranose-containing gadolinium(III) complex [Gd(DOTA-FPG)(H2O)] was designed, synthesized, and characterized as being potentially suitable for a bioactivated MRI contrast agent. The 17O NMR experiments were conducted to estimate the water exchange rate kex298 and rotational correlation timeτR298. The kex298 value of [Gd(DOTA-FPG)(H2O)] is similar to that of [Gd(DO3A-bz-NO 2)(H2O)]. The rotational correlation time value of [Gd(DOTA-FPG)(H2O)] is dramatically longer than that of [Gd(DOTA)(H2O)]- Relaxometric studies show that the percentage change in the T1 value of [Gd(DOTA-FPG)(H2O)] decreases dramatically in the presence of β-galactosidase and human serum albumin. The T1 change percentage of [Gd(DOTA-FPG)(H2O)] (60%) is significantly higher than those of Egad and gadolinium(III)-1-(4-(2-(1- (4,7,10-triscarboxymethyl-(1,4,7,10-tetraazacyclododecyl)))- ethylcarbamoyloxymethyl)-2-nitrophenyl)-β-D-glucopyronuronate. The signal intensity of the MR image for [Gd(DOTA-FPG)(H2O)] in the presence of human serum albumin and β-galactosidase (2670 ± 210) is significantly higher than that of [Gd(DOTA-FPG)(H2O)] in the sodium phosphate buffer solution (1490 ± 160). In addition, the MR images show a higher-intensity enhancement in CT26/β-gal tumor with β-galactosidase gene expression but not for the CT26 tumor without β-galactosidase gene expression. We conclude that [Gd(DOTA-FPG)(H2O)] is a suitable candidate for a bioactivated MRI contrast agent in tracing gene expression.