This work aims to further understand the grain size effects on individual slip/twining activities at grain scale and their correlation with the tensile behavior, for a Mg-5Y sheet with mean grain size range of 3–130 μm, based on slip trace/electron backscatter diffraction (EBSD) analysis. The correlation between tensile yield strength and the corresponding grain size fitted well with the Hall-Petch relationship, i.e., the k value was 191 MPa·μm1/2, with an R2 of 0.96. According to the identified 1004 sets of slip traces in 2477 grains, basal <a> slip was always dominant and tended to increase (from 47.2% to 71.5%) with grain size increased from 3 to 85 μm. Meanwhile, the percentage of pyramidal II <c+a> slip exhibited a marked decline from 34.5% to 8.9%, while that of prismatic <a> and pyramidal I <c+a> slip changed slightly. The decreased pyramidal II <c+a> slip activity was consistent with the decreased tensile yield strength as grain size increased. The twinning activity was limited and insensitive to grain size. In addition, the geometrically necessary dislocation (GND) density was increased with increasing grain size. Based on the slip activity statistics, the critical resolved shear stress (CRSS) ratios were estimated, which exhibited a significant grain size effect. Specifically, CRSSpris/CRSSbas showed negative grain size dependence, while the opposite was true for CRSSpyrIICA/CRSSbas. This study provides valuable grain-scale experimental evidence for the grain size effect on individual slip activity and CRSS ratios, which contributes to a better understanding of the Hall-Petch relationship in Mg-Y alloy.