Plant color is a key feature for estimating parameters of the plant grown under different conditions using remote sensing images. In this case, the variation in plant color should be only due to the influence of the growing conditions and not due to external confounding factors like a light source. Hence, the impact of the light source in plant color should be alleviated using color calibration algorithms. This study aims to develop an efficient, robust, and cutting-edge approach for automatic color calibration of three-band (red green blue: RGB) images. Specifically, we combined the k-means model and deep learning for accurate color calibration matrix (CCM) estimation. A dataset of 3150 RGB images for oilseed rape was collected by a proximal sensing technique under varying illumination conditions and used to train, validate, and test our proposed framework. Firstly, we manually derived CCMs by mapping RGB color values of each patch of a color chart obtained in an image to standard RGB (sRGB) color values of that chart. Secondly, we grouped the images into clusters according to the CCM assigned to each image using the unsupervised k-means algorithm. Thirdly, the images with the new cluster labels were used to train and validate the deep learning convolutional neural network (CNN) algorithm for an automatic CCM estimation. Finally, the estimated CCM was applied to the input image to obtain an image with a calibrated color. The performance of our model for estimating CCM was evaluated using the Euclidean distance between the standard and the estimated color values of the test dataset. The experimental results showed that our deep learning framework can efficiently extract useful low-level features for discriminating images with inconsistent colors and achieved overall training and validation accuracies of 98.00% and 98.53%, respectively. Further, the final CCM provided an average Euclidean distance of 16.23 ΔE and outperformed the previously reported methods. This proposed technique can be used in real-time plant phenotyping at multiscale levels.