The objective of this study was to quantify the effects of cyclic irrigation on growth and physiology of container-grown conifer species in pot-in-pot (PIP) production in the upper Midwest. Trees of four conifer species (Picea glauca var. densata, Picea pungens, Abies fraseri, and Pinus strobus) were grown in 25-L containers and assigned to one of four combinations of irrigation rate (low or high) and daily irrigation cycle frequency (one or four). Irrigation rates were based on common nursery practice in the region (2 cm rainfall equivalent/day) and one-half the standard rate (1 cm rainfall equivalent/day). Cyclic irrigation increased relative height growth and relative caliper growth of Pinus strobus by over 80% and 35%, respectively, compared with once-daily irrigation. The high-rate irrigation increased relative caliper growth of Picea pungens by 40% compared with the low rate. The effects of irrigation regime on needle- or shoot-level gas exchange varied by species and date of measurement. Carbon isotope discrimination (Δ13C) of needle and wood tissue was positively correlated (r ≥ 0.64, P < 0.001) with needle conductance to water vapor (gwv) and negatively correlated (r ≤ −0.60, P< 0.001) with intrinsic water use efficiency (WUEi). Carbon isotope discrimination of wood and needle tissue decreased with the low irrigation rate, indicating increased WUEi associated with reduced gwv. Cyclic irrigation had relatively little effect on Δ13C except for Pinus strobus. Our findings suggest that carbon isotope composition of wood and needle tissue provides a sensitive and accurate representation of plant response to varying moisture availability. From a water management perspective, identifying optimal irrigation rates appears to be more important than number of daily cycles for these crops grown in the midwestern United States.