Self-guided web-based interventions have the potential of addressing help-seeking barriers and symptoms common among university students, such as depression and anxiety. Unfortunately, self-guided interventions are also associated with less adherence, implicating motivation as a potential moderator for adherence and improvement for such interventions. Previous studies examining motivation as a moderator or predictor of improvement on web-based interventions have defined and measured motivation variably, producing conflicting results. This secondary analysis of data from a randomized controlled trial aimed to examine constructs of motivation as moderators of improvement for a self-guided 8-week web-based intervention in university students (N=1607). Tested moderators included internal motivation, external motivation, and confidence in treatment derived from the Treatment Motivation Questionnaire. The primary outcome was an improvement in depression and anxiety measured by the Depression Anxiety Stress Scale-21. Piecewise linear mixed effects models showed that internal motivation significantly moderated symptom change for the intervention group (t1504=-2.94; P=.003) at average and high (+1 SD) motivation levels (t1507=-2.28; P=.02 and t1507=-4.05; P<.001, respectively). Significant results remained even after controlling for baseline severity. The results showed that confidence in treatment did not significantly moderate symptom change for the intervention group (t1504=1.44; P=.15). In this sample, only internal motivation was positively correlated with service initiation, intervention adherence, and intervention satisfaction. The combination of a web-based intervention and high or moderate internal motivation resulted in greater improvement in the total Depression Anxiety Stress Scale-21 score. These findings highlight the importance of conceptually differentiating motivation-related constructs when examining moderators of improvement. The results suggest that the combination of a web-based intervention and high or moderate internal motivation results in greater improvement. These findings highlight the importance of conceptually differentiating motivation-related constructs when examining moderators of improvement. To better understand the moderating role of internal motivation, future research is encouraged to replicate these findings in diverse samples as well as to examine related constructs such as baseline severity and adherence. Understanding these characteristics informs treatment strategies to maximize adherence and improvement when developing web-based interventions as well as allows services to be targeted to individuals likely to benefit from such interventions. ClinicalTrials.gov NCT04361045; https://clinicaltrials.gov/study/NCT04361045.