Given the increasing prevalence of web technology, web-based mathematics environments have been increasingly widely used in mathematics education for the past two decades. The COVID-19 pandemic has led to an urgent transition from traditional mathematics instruction (TMI) to web-based mathematics instruction (WBMI) at all levels of mathematics education. At this point, it is crucial to scrutinize the effects of WBMI on K-16 students’ mathematics learning comprehensively. This meta-analysis research contained a total of 63 studies with 115 effect sizes, which aimed to investigate the effectiveness of WBMI on K-16 students’ mathematics learning by incorporating potential moderators, namely mathematics topics, mathematical content standards, feedback status, type of instructional features, age (i.e., grade level), and assessment methods. Based on findings, WBMI has a significantly strong effect on K-16 students’ mathematics learning (
g
= 1.10,
p
= 0.01, 95% CI [0.95, 1.27]). Moderator analyses reveal that the effect sizes of WBMI on K-16 students’ mathematics learning varied significantly depending on all these potential moderators. Additionally, higher-level mathematical concepts, statistics and probability, WBMI with providing feedback, tutorial systems, undergraduate students, and traditional paper-pencil assessment are the strongest moderators in their context. The most notable results of this research are that WBMI is significantly more effective on students’ mathematics learning than TMI, while even in the context of WBMI, traditional paper-pencil assessment is significantly more effective than online assessment. This meta-analytic research provides a comprehensive and up-to-date perspective on the effectiveness of WBMI on K-16 students’ mathematics learning.