Purpose <p>T1rho imaging showed potential applications in cancer imaging but little research explored the underlying biological processes that contribute to the T1rho values in cancer. This study aimed to investigate the potential associations between quantitative imaging biomarkers from T1rho imaging and the well-established diffusion weighted imaging (DWI), with tumour-stromal, immunohistochemical (IHC), and tumour-infiltration-lymphocytes (TIL) biomarkers in nasopharyngeal carcinoma (NPC).</p> Methods <p>Pre-treatment T1rho and DWI imaging of primary NPCs were performed in 50 prospectively recruited patients. The mean T1rho and apparent diffusion coefficient (ADC) of NPC were obtained and correlated with tumour-stromal, IHC, TIL biomarkers using the Pearson Correlation test and the coefficients (R) were calculated.</p> Results <p>The mean T1rho values negatively correlated with collagenous stroma-lymphoid stroma (<i>R</i>=-0.314, <i>p</i> = 0.03) and positively correlated with percentage of tumour cells positive for Ki-67 (<i>R</i> = 0.402, <i>p</i> &lt; 0.01), but there were no associations between T1rho values and the other tumour-stromal, IHC or TIL biomarkers (<i>p</i> = 0.16–0.98) or between ADC values and any of these biomarkers (<i>p</i> = 0.07–0.82).</p> Conclusion <p>Our results showed the possible underlying biological mechanisms of T1rho imaging in head and neck cancer. T1rho imaging negatively correlated with the ratio of collagenous to lymphoid stroma, and positively correlated with tumour cell proliferation, which are both known to be predictors of outcome, suggesting that T1rho imaging may have a valuable role in head and neck cancer imaging. As this is a preliminary study with small sample size, further studies are encouraged to validate our findings.</p>