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The aim of this study was to investigate the effects of postnatal choline supplementation on neurochemical and behavioral parameters in preweanling BALB/cByJ mice. Mouse pups were injected daily subcutaneously with choline chloride (0.85 mM/g body weight) from Postnatal Day (PND) 1 to PND 16. Pups performed a passive avoidance (PA) learning task on PND 17–18 and a 30-min locomotor activity test on PND 19. The choline treatment affected retention of the PA task on PND 18. The treatment also increased locomotor activity in females, but not in males, on PND 19. Choline acetyltransferase (ChAT) enzymatic activity was measured on PND 20 and revealed that choline administration in the first 2 weeks of postnatal life selectively affects male pups. Choline's effect, as seen in previous rat experiments, was to decrease ChAT activity in the hippocampal region.