Abstract Background Accurate measurement of the components of physical activity during pregnancy can aid in our understanding of the dose response relationships between physical activity and corresponding perinatal outcomes. The aim of this study was to develop and evaluate a one-week recall questionnaire to assess moderate to vigorous physical activity during pregnancy. Methods To assess concurrent-related validity, 177 pregnant women (median 18 weeks' gestation, interquartile range (IQR) 15 -23) kept a structured diary and wore an accelerometer (Actigraph) for one week. At the conclusion of the week, they completed the Pregnancy Infection and Nutrition 3 (PIN3) physical activity questionnaire over the telephone. To assess evidence for test-retest reliability, 109 pregnant women (median 19 weeks' gestation, IQR 18-27) completed the questionnaire twice over the telephone, within 48 hours apart, recalling the same two time periods. Spearman correlation coefficients (SCC) and intraclass correlation coefficients (ICC) were used to assess evidence for validity and reliability, respectively. Results Comparison of the questionnaire to the structured diary was moderate to substantial (SCC 0.47 to 0.69) for several measures of moderate or vigorous physical activity using either perceived or absolute intensity. Comparison of moderate to vigorous physical activity from the questionnaire (absolute intensity using MET-hours/week) to the accelerometer ranged from 0.12 to 0.23 using SCC for absolute intensity (MET-hours/week) and 0.28 to 0.34 using relative intensity (hours/week) (n = 177). Test-retest reliability was moderate to almost perfect for moderate to vigorous physical activity, with the ICC ranging from 0.56 to 0.82 for both perceived and absolute intensities. Conclusions The PIN3 one-week recall questionnaire assessed moderate to vigorous physical activity in the past week with evidence for reliability and validity.