Science motivation is an important factor that directly influences students' science learning. Numerous studies have been undertaken to develop and validate questionnaire items for measuring students' motivation in science learning. This study is the first longitudinal examination of the Chinese version of Science Motivation Questionnaire II (SMQ II-C) in a Chinese cultural context. Using two waves of surveys, we evaluated its internal structure validity and criterion-related validity. Results showed that at each time point, scores were internally consistent and the hypothesized five-factor model was confirmed as the best model fit for the data. Results of multigroup invariance revealed the structure of the SMQ II-C was equivalent within gender subgroups. Furthermore, the present study added longitudinal invariance evidence of the SMQ II-C by sampling two-time points. Overall, this study suggests the SMQ II-C is a robust instrument for evaluating Chinese high school students' motivation to learn science. Furthermore, boys yielded higher scores than girls among all five subscales of science motivation and significant gender differences were observed in both waves. Implications and limitations of these results are discussed.