The human rhinovirus (HRV) 3C and 2A proteases (3C(pro) and 2A(pro), respectively) are critical in HRV infection, as they are required for viral polyprotein processing as well as proteolysing key host factors to facilitate virus replication. Early in infection, 3C(pro) is present as its precursor 3CD, which, although the mechanism of subcellular targeting is unknown, is found in the nucleus as well as the cytoplasm. In this study, we use transfected and infected cell systems to show that 2A(pro) activity is required for 3CD nuclear localization. Using green fluorescent protein (GFP)-tagged forms of 3C(pro), 3D, and mutant derivatives thereof, we show that 3C(pro) is located in the cytoplasm and the nucleus, whereas 3CD and 3D are localized predominantly in the cytoplasm, implying that 3D lacks nuclear targeting ability and that 3C(pro) activity within 3CD is not sufficient to allow the larger protein into the nucleus. Importantly, by coexpressing mCherry-2A(pro) fusion proteins, we demonstrate formally that 2A(pro) activity is required to allow HRV 3CD access to the nucleus. In contrast, mCherry-3C(pro) is insufficient to allow 3CD access to the nucleus. Finally, we confirm the relevance of these results to HRV infection by demonstrating that nuclear localization of 3CD correlates with 2A(pro) activity and not 3C(pro) activity, which is observed only later in infection. The results thus define the temporal activities of 2A(pro) and 3CD/3C(pro) activities in HRV serotype16 infection. IMPORTANCE: The human rhinovirus genome encodes two proteases, 2A and 3C, as well as a precursor protease, 3CD. These proteases are essential for efficient virus replication. The 3CD protein is found in the nucleus early during infection, though the mechanism of subcellular localization is unknown. Here we show that 2A protease is required for this localization, the 3C protease activity of 3CD is not sufficient to allow 3CD entry into the nucleus, and 3D lacks nuclear targeting ability. This study demonstrates that both 2A and 3C proteases are required for the correct localization of proteins during infection and defines the temporal regulation of 2A and 3CD/3C protease activities during HRV16 infection.