The superficial layers of primary visual cortex, unlike layer 4, have an extensive network of long-range horizontal connections linking sites of similar orientation preference. To identify possible functional consequences of this distinct anatomy, we compared the receptive field properties of layers 2/3 and 4 neurons in tree shrew primary visual cortex with electrophysiological recordings. We found that elongated receptive fields, strong orientation tuning, and length summation (properties predicted by the anatomy of the horizontal connections) are present in layer 2/3 neurons, but not in layer 4 neurons. We further characterized the summation fields of layer 2/3 neurons and found axis and orientation-specific facilitation that matched the distribution of horizontal connections. The functional signature of horizontal connections was also evident in the population response of layer 2/3 neurons; the intrinsic signal activation pattern elicited by an array of collinear Gabor elements was significantly stronger than that elicited by a noncollinear array. Furthermore, our results showed that this enhancement of population response was achieved without compromising spatial resolution along the collinear axis, providing stimulus-specific facilitation without filling in between stimuli. Taken together, these results suggest that horizontal connections play a significant role in shaping the visual responses of layer 2/3 neurons.